Methods of making chimeric antigen receptor-expressing cells

ABSTRACT

The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), and compositions and reaction mixtures comprising the same.

This application claims priority to U.S. Ser. No. 62/097,375 filed Dec.29, 2014 and U.S. Ser. No. 62/133,137 filed Mar. 13, 2015, the contentsof which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 23, 2015, isnamed N2067-706710_SL.txt and is 243,586 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to methods of making immuneeffector cells (e.g., T cells, NK cells) engineered to express aChimeric Antigen Receptor (CAR), and compositions comprising the same.

BACKGROUND OF THE INVENTION

Adoptive cell transfer (ACT) therapy with autologous T-cells, especiallywith T-cells transduced with Chimeric Antigen Receptors (CARs), hasshown promise in several hematologic cancer trials.

SUMMARY OF THE INVENTION

The present disclosure pertains to methods of making immune effectorcells (e.g., T cells, NK cells) that can be engineered to express a CAR,and compositions comprising the same.

Accordingly, in one aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells) that canbe engineered to express a CAR, the method comprising providing apopulation of immune effector cells (e.g., T cells), removing Tregulatory cells, e.g., CD25+ T cells, from the population, to therebyprovide a population of T regulatory-depleted cells, e.g., CD25+depleted cells, that are suitable for expression of a CAR.

In one embodiment, the population of T regulatory-depleted cellscontains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+cells.

In one embodiment, the population of immune effector cells are cells ofa subject having cancer, e.g., a subject having a CD25 expressing cancersuch as, e.g., chronic lymphocytic leukemia (CLL). In one embodiment,the population of T regulatory-depleted cells contains less than 50%,40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and lessthan 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of tumorcells.

In one embodiment, the population of immune effector cells areautologous to the subject who the cells will be administered to fortreatment. In one embodiment, the population of immune effector cellsare allogeneic to the subject who the cells will be administered to fortreatment.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using an anti-CD25 antibody, or fragmentthereof, or a CD25-binding ligand, e.g. IL-2. In one embodiment, theanti-CD25 antibody, or fragment thereof, or CD25-binding ligand isconjugated to a substrate, e.g., a bead, or is otherwise coated on asubstrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, orfragment thereof, is conjugated to a substrate as described herein. Inone embodiment, the T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody molecule, or fragmentthereof.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depletion reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL.

In one embodiment, the population of T regulatory-depleted cells, e.g.,CD25+ depleted cells, are suitable for expression of a CAR describedherein, e.g., a CD19 CAR described herein. In one embodiment, thepopulation of immune effector cells are obtained from a subject having ahaematological cancer, e.g., a leukemia, e.g., chronic lymphocyticleukemia (CLL), acute lymphocytic leukemia (ALL), or a lymphoma, e.g.,mantle cell lymphoma (MCL) or Hodgkin lymphoma (HL). In one embodiment,the population of T regulatory-depleted cells contains less than 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the leukemia cells, e.g., CLLcells, ALL cells, or lymphoma cells, e.g., MCL cells or HL cells. In oneembodiment, the population of immune effector cells are obtained from asubject having CLL, and the population of T regulatory-depleted cells,e.g., CD25+ depleted cells, contains less than 30%, 25%, 20%, 15%, 10%,5%, 4%, 3%, 2%, 1% of the leukemia cells, e.g., CLL cells and aresuitable for expression of a CD19 CAR described herein. In oneembodiment, the population of T regulatory-depleted cells contains lessthan 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 15%, 10%,5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CD25 expressing tumor cells,e.g., CLL cells. In one embodiment, the population of Tregulatory-depleted cells contains less than 10%, 5%, 4%, 3%, 2%, 1% ofCD25+ cells and less than 10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g.,CD25 expressing tumor cells, e.g., CLL cells.

In one embodiment, the population of immune effector cells are T cellsisolated from peripheral blood lymphocytes. In an embodiment, thepopulation of T cells are obtained by lysing the red blood cells and/orby depleting the monocytes. In an embodiment, the population of T cellsis isolated from peripheral lymphocytes using, e.g., a method describedherein.

In one embodiment, the population of immune effector cells can beobtained from a blood sample from a subject, e.g., obtained byapheresis. In one embodiment, the cells collected by apheresis arewashed to remove the plasma fraction and, optionally, the cells areprovided in an appropriate buffer or media for subsequent processingsteps. In one embodiment, the cells are washed with a buffer such as,e.g., phosphate buffered saline (PBS). In an embodiment, the cells arewashed in a wash solution that lacks one or more divalent cation such ascalcium and magnesium, e.g., lacks both calcium and magnesium. In oneembodiment, the cells are washed in a buffer that has substantially nodivalent cations.

In one embodiment, the method further comprises removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11b, to thereby provide a population of T regulatory depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

In one embodiment, the method further comprises removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of (e.g., 2 or 3 of) PD1+cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population ofT regulatory depleted, e.g., CD25+ depleted cells, and check pointinhibitor depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells.In embodiments, PD1+ cells and LAG3+ cells are removed; PD1+ cells andTIM3+ cells are removed; or LAG3+ and TIM3+ cells are removed. In oneembodiment, check point inhibitor expressing cells are removedsimultaneously with the T regulatory, e.g., CD25+ cells. For example, ananti-CD25 antibody, or fragment thereof, and an anti-check pointinhibitor antibody, or fragment thereof, can be attached to the samebead which can be used to remove the cells, or an anti-CD25 antibody, orfragment thereof, and the anti-check point inhibitor antibody, orfragment there, can be attached to separate beads, a mixture of whichcan be used to remove the cells. In other embodiments, the removal of Tregulatory cells, e.g., CD25+ cells, and the removal of the check pointinhibitor expressing cells is sequential, and can occur, e.g., in eitherorder.

In one embodiment, the population of cells to be removed are neither theregulatory T cells or tumor cells, but cells that otherwise negativelyaffect the expansion and/or function of CART cells, e.g. cellsexpressing CD14, CD11b, CD33, CD15, or other markers expressed bypotentially immune suppressive cells. In one embodiment, such cells areenvisioned to be removed concurrently with regulatory T cells and/ortumor cells, or following said depletion, or in another order.

In one embodiment, the method further comprises removing cells from thepopulation which express CD14, to thereby provide a population of Tregulatory-depleted, e.g., CD25+ depleted cells, and CD14+ depletedcells. In one embodiment, CD14+ cells are removed simultaneously withthe T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody,or fragment thereof, and an anti-CD14 antibody, or fragment thereof, canbe attached to the same bead which can be used to remove the cells; oran anti-CD25 antibody, or fragment thereof, and the anti-CD14 antibody,or fragment thereof, can be attached to separate beads, a mixture ofwhich can be used to remove the cells. In other embodiments, the removalof T regulatory cells, e.g., CD25+ cells, and the removal of the CD14+cells is sequential, and can occur, e.g., in either order. In oneembodiment, the CD14+ cells are removed using a CD14 antibody moleculeor fragment thereof.

In one embodiment, the population of immune effector cells provided havebeen selected based upon the expression of one or more markers (e.g., 2,3, 4, 5, 6, 7, or more markers), e.g., CD3, CD28, CD4, CD8, CD27, CD127,CD45RA, and CD45RO, e.g., the provided population of immune effectorcells (e.g., T cells) are CD3+ and/or CD28+.

In one embodiment, the method further comprises obtaining a populationof immune effector cells, e.g., T cells, enriched for the expression ofone or more markers (e.g., 2, 3, 4, 5, 6, 7, or more markers), e.g.,CD3, CD28, CD4, CD8, CD27, CD127, CD45RA, and CD45RO. In an embodiment,population of immune effector cells are enriched for CD3+ and/or CD28+cells. For example, T cells isolated by incubation withanti-CD3/anti-CD28 conjugated beads are obtained. In one embodiment, themethod further comprises selecting cells from the population of Tregulatory-depleted cells, e.g., CD25+ depleted cells, which express oneor more markers (e.g., 2, 3, 4, 5, or more markers), e.g., CD3, CD28,CD4, CD8, CD45RA, and CD45RO.

In one embodiment, the method further comprises activating thepopulation of T regulatory depleted cells, e.g., CD25+ depleted cells,e.g., by a method described herein.

In one embodiment, the method further comprises transducing a cell fromthe population of T regulatory-depleted cells, e.g., the population ofCD25+ depleted cells, with a vector comprising a nucleic acid encoding aCAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein. Inone embodiment, the vector is selected from the group consisting of aDNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or aretrovirus vector. In one embodiment, the cell from the population of Tregulatory-depleted cells, e.g., the population of CD25+ depleted cells,is transduced with a vector once, e.g., within one day after populationof immune effector cells are obtained from a blood sample from asubject, e.g., obtained by apheresis.

In one embodiment, the method further comprises generating a populationof RNA-engineered cells transiently expressing exogenous RNA from thepopulation of T regulatory-depleted cells, e.g., the population of CD25+depleted cells. The method comprises introducing an in vitro transcribedRNA or synthetic RNA into a cell from the population, where the RNAcomprises a nucleic acid encoding a CAR, e.g., a CAR described herein,e.g., a CD19 CAR described herein.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, e.g., a CD19 CAR described herein, areexpanded, e.g., by a method described herein. In one embodiment, thecells are expanded in culture for a period of several hours (e.g., about2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment,the cells are expanded for a period of 3 to 9 days. In one embodiment,the cells are expanded for a period of 4 to 9 days. In one embodiment,the cells are expanded for a period of 8 days or less, e.g., 7, 6, 5, 4,or 3 days. In one embodiment, the cells, e.g., a CD19 CAR cell describedherein, are expanded in culture for 3 or 4 days, and the resulting cellsare more potent than the same cells expanded in culture for 9 days underthe same culture conditions. In one embodiment, the cells, e.g., a CD19CAR cell described herein, are expanded in culture for 5 days, and theresulting cells are more potent than the same cells expanded in culturefor 9 days under the same culture conditions. Potency can be defined,e.g., by various T cell functions, e.g. proliferation, target cellkilling, cytokine production, activation, migration, or combinationsthereof. In one embodiment, the cells, e.g., a CD19 CAR cell describedherein, expanded for 3 or 4 days show at least a one, two, three or fourfold increase in cells doublings upon antigen stimulation as compared tothe same cells expanded in culture for 9 days under the same cultureconditions. In one embodiment, the cells, e.g., a CD19 CAR celldescribed herein, expanded for 5 days show at least a one, two, three orfour fold increase in cells doublings upon antigen stimulation ascompared to the same cells expanded in culture for 9 days under the sameculture conditions. In one embodiment, the cells, e.g., the cellsexpressing a CD19 CAR described herein, are expanded in culture for 3 or4 days, and the resulting cells exhibit higher proinflammatory cytokineproduction, e.g., IFN-γ and/or GM-CSF levels, as compared to the samecells expanded in culture for 9 days under the same culture conditions.In one embodiment, the cells, e.g., the cells expressing a CD19 CARdescribed herein, are expanded in culture for 5 days, and the resultingcells exhibit higher proinflammatory cytokine production, e.g., IFN-γand/or GM-CSF levels, as compared to the same cells expanded in culturefor 9 days under the same culture conditions. In one embodiment, thecells, e.g., a CD19 CAR cell described herein, expanded for 3 or 4 daysshow at least a one, two, three, four, five, ten fold or more increasein pg/ml of proinflammatory cytokine production, e.g., IFN-γ and/orGM-CSF levels, as compared to the same cells expanded in culture for 9days under the same culture conditions. In one embodiment, the cells,e.g., a CD19 CAR cell described herein, expanded for 5 days show atleast a one, two, three, four, five, ten fold or more increase in pg/mlof proinflammatory cytokine production, e.g., IFN-γ and/or GM-CSFlevels, as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g., aCD19 CAR cell described herein, expanded for 3, 4, or 5 days show atleast as high cytokine production in pg/ml, or at least a one, two,three, four, five, ten fold or more increase in pg/ml of cytokineproduction, e.g., IL2, IFN-gamma, GM-CSF, TNF-alpha, IL-1b, IL4, IL5,IL6, IL8, or IL10, levels, as compared to the same cells expanded inculture for 9 days under the same culture conditions.

In one embodiment, beads (e.g., CD3/28-stimulatory beads) are removedfrom the cells by mechanical disruption. In an embodiment, mechanicaldisruption comprises passage (e.g., repeat passage) cells through apipette tip, e.g., a narrow bore pipette tip. In some embodiment, thecells are passed through one or more (e.g., 2, 3, 4, or 5 or more)tubes, e.g., narrow bore tubes. In one embodiment, the tubes are part ofa closed cell culture system. In some embodiments, the interior diameterof the pipet tip or tubes is less than about 1 mm, 0.9 mm, 0.8 mm, 0.7mm, 0.5 mm, 0.4 mm, 0.3 mm, or 0.2 mm, and optionally the diameter isgreater than 0.1 mm, 0.2 mm, 0.3 mm, or 0.4 mm.

In one embodiment, the cells, e.g., CAR cells described herein, e.g.,CD19 CAR cells described herein, expanded for 3, 4, or 5 days, have anincreased proportion of Tem cells, Tcm cells, or both Tem and Tcm cells,compared to as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g.,CAR cells described herein, e.g., CD19 CAR cells described herein,expanded for 3, 4, or 5 days, have an increased proportion of Tem cells,Teff cells, or both Tem and Teff cells, compared to as compared to thesame cells expanded in culture for 9 days under the same cultureconditions. In one embodiment, a population of cells, e.g., CD19CARcells described herein, expanded for 3, 4, or 5 days, has a percentageof Tnaive like cells (out of Tnaive like, Teff, and Tcm cells) of atleast about 20%, 25%, 30%, 35%, 40%, 45%, or 50%, and optionally up toabout 40% or 50%. In one embodiment, a population of cells, e.g.,CD19CAR cells described herein, expanded for 3, 4, or 5 days, has apercentage of Tem cells (out of Tnaive like, Teff, and Tcm cells) of atleast about 10%, 15%, or 20%, and optionally up to about 15% or 20%. Inone embodiment, a population of cells, e.g., CD19CAR cells describedherein, expanded for 3, 4, or 5 days, has a percentage of (Tnaivelike+Tem) cells (out of Tnaive like, Teff, and Tcm cells) of at leastabout 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, andoptionally up to about 50% or 70%.

In one embodiment, the cells, e.g., CD19 CAR cells described herein,expanded for 3, 4, or 5 days, when administered at a dose of 0.5×10⁶cells in the NALM6 assay of FIG. 31A have an activity greater than orequal to a dose of 1×10⁶, 10.5×10⁶, 2×10⁶, 2.5×10⁶, 3×10⁶, 4×10⁶, or5×10⁶ of the same cells expanded in culture for 9 days under the sameculture conditions. In some embodiments activity is measured at 1, 2, 3,4, 5, or 6 weeks.

In one embodiment, the cells are expanded by culturing the cells in thepresence of an agent that stimulates a CD3/TCR complex associated signaland a ligand that stimulates a costimulatory molecule on the surface ofthe cells, e.g., as described herein. In one embodiment, the agent is abead conjugated with anti-CD3 antibody, or a fragment thereof, and/oranti-CD28 antibody, or a fragment thereof.

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that may, optionally, contain one or more (e.g.,2, 3, 4, or 5 or more) factor for proliferation and/or viability,including serum (e.g., fetal bovine or human serum), interleukin-2(IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21,TGFβ, and TNF-α or any other additives for the growth of cells.

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more (e.g., 2, 3, 4, or 5or more) interleukins that result in at least a 200-fold (e.g.,200-fold, 250-fold, 300-fold, 350-fold) increase in cells over a 14 dayexpansion period, e.g., as measured by a method described herein such asflow cytometry. In one embodiment, the cells are expanded in thepresence of IL-15 and/or IL-7 (e.g., IL-15 and IL-7).

In one embodiment, the cells are cryopreserved after the appropriateexpansion period. In one embodiment, the cells are cryopreservedaccording to a method described herein. In one embodiment, the expandedcells are cryopreserved in an appropriate media, e.g., an infusiblemedia, e.g., as described herein.

In one embodiment, the method further comprises contacting thepopulation of immune effector cells with a nucleic acid encoding atelomerase subunit, e.g., hTERT. In an embodiment, the nucleic acid isDNA or RNA.

In another aspect, the disclosure features a reaction mixture comprisinga population of T regulatory-depleted cells containing less than 50%,40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells. In oneembodiment, the reaction mixture comprises a population of Tregulatory-depleted cells containing less than 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25 expressing tumor cells,e.g., CLL cells. In one embodiment, the population of cells containsless than 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 15%,10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CD25 expressing tumorcells, e.g., CLL cells. In one embodiment, the population of cellscontains less than 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CD25 expressing tumorcells, e.g., CLL cells.

In one embodiment, the reaction mixture comprises a population of Tregulatory-depleted cells containing less than 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of a checkpoint inhibitorexpressing cells, e.g., a PD1+ cells, LAG3+ cells, or TIM3+ cells. Thereaction mixture may further comprise a buffer or other reagent, e.g., aPBS containing solution.

In one embodiment, the reaction mixture comprises a population of Tregulatory-depleted cells containing less than 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD14+ cells. The reactionmixture may further comprise a buffer or other reagent, e.g., a PBScontaining solution.

In one embodiment, the reaction mixture can further comprise an agentthat activates and/or expands to cells of the population, e.g., an agentthat stimulates a CD3/TCR complex associated signal and/or a ligand thatstimulates a costimulatory molecule on the surface of the cells, e.g.,as described herein. In one embodiment, the agent is a bead conjugatedwith anti-CD3 antibody, or a fragment thereof, and/or anti-CD28antibody, or a fragment thereof.

In one embodiment, the reaction mixture further comprises one or more(e.g., 2, 3, 4, or 5) factor for proliferation and/or viability,including serum (e.g., fetal bovine or human serum), interleukin-2(IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21,TGFβ, and TNF-α or any other additives for the growth of cells. In oneembodiment, the reaction mixture further comprises IL-15 and/or IL-7.

In one embodiment, a plurality of the cells of the population in thereaction mixture comprise a nucleic acid molecule, e.g., a nucleic acidmolecule described herein, that comprises a CAR encoding sequence, e.g.,a CD19 CAR encoding sequence, e.g., as described herein.

In one embodiment, a plurality of the cells of the population in thereaction mixture comprise a vector comprising a nucleic acid sequenceencoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR describedherein. In one embodiment, the vector is a vector described herein,e.g., a vector selected from the group consisting of a DNA, a RNA, aplasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

In one embodiment, the reaction mixture further comprises acryoprotectant or stabilizer such as, e.g., a saccharide, anoligosaccharide, a polysaccharide and a polyol (e.g., trehalose,mannitol, sorbitol, lactose, sucrose, glucose and dextran), salts andcrown ethers. In one embodiment, the cryoprotectant is dextran.

In another aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells) engineeredto express a CAR, the method comprising providing a population of immuneeffector cells (e.g., T cells), wherein a plurality of the immuneeffector cells comprise a nucleic acid encoding a CAR, e.g., a CARdescribed herein, e.g., a CD19 CAR described herein, and expanding thecells of the population in the presence of one or more (e.g., 2, 3, 4,or 5) interleukin that result in at least a 200-fold (e.g., 200-fold,250-fold, 300-fold, 350-fold) increase in cells over a 14 day expansionperiod, e.g., as measured by a method described herein such as flowcytometry. In one embodiment, the cells of the population are expandedin the presence of IL-15 and/or IL-7, e.g., IL-15 and IL-7.

In one embodiment, the cells are expanded in culture for a period ofseveral hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours)to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14days). In one embodiment, the cells are expanded for a period of 4 to 9days. In one embodiment, the cells are expanded for a period of lessthan 8 days, e.g., 7, 6 or 5 days. In one embodiment, the cells areexpanded in culture for 5 days, and the resulting cells are more potentthan the same cells expanded in culture for 9 days under the sameculture conditions. Potency can be defined, e.g., by various T cellfunctions, e.g. proliferation, target cell killing, cytokine production,activation, migration, or combinations thereof. In one embodiment, thecells, e.g., a CD19 CAR cell described herein, expanded for 5 days showat least a one, two, three or four fold increase in cells doublings uponantigen stimulation as compared to the same cells expanded in culturefor 9 days under the same culture conditions. In one embodiment, thecells, e.g., the cells expressing a CD19 CAR described herein, areexpanded in culture for 5 days, and the resulting cells exhibit higherproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells, e.g., a CD19 CARcell described herein, expanded for 5 days show at least a one, two,three, four, five, ten fold or more increase in pg/ml of proinflammatorycytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared tothe same cells expanded in culture for 9 days under the same cultureconditions.

In one embodiment, the cells are expanded by culturing the cells in thepresence of an agent that stimulates a CD3/TCR complex associated signaland a ligand that stimulates a costimulatory molecule on the surface ofthe cells, e.g., as described herein. In one embodiment, the agent is abead conjugated with anti-CD3 antibody, or a fragment thereof, and/oranti-CD28 antibody, or a fragment thereof.

In one embodiment, the provided population of immune effector cells is apopulation of T regulatory-depleted cells containing less than 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells. In oneembodiment, the provided population of immune effector cells is apopulation of T regulatory-depleted cells containing less than 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25 expressingtumor cells, e.g., CLL cells. In one embodiment, the provided populationof cells contains less than 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cellsand less than 15%, 10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CD25expressing tumor cells, e.g., CLL cells. In one embodiment, the providedpopulation of cells contains less than 10%, 5%, 4%, 3%, 2%, 1% of CD25+cells and less than 10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CD25expressing tumor cells, e.g., CLL cells.

In one embodiment, the provided population of immune effector cells is apopulation of T regulatory-depleted cells containing less than 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of a checkpointinhibitor expressing cells, e.g., a PD1+ cells, LAG3+ cells, or TIM3+cells.

In one embodiment, the provided population of immune effector cells is apopulation of T regulatory-depleted cells containing less than 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD14+ cells.

In one embodiment, the method further comprises, prior to expansion,removing T regulatory cells, e.g., CD25+ T cells, from the population,to thereby provide a population of T regulatory-depleted cells, e.g.,CD25+ depleted cells to be expanded. In one embodiment, the T regulatorycells, e.g., CD25+ cells, are removed by a method described herein.

In one embodiment, the method further comprises, prior to expansion,removing T regulatory cells, e.g., CD14+ cells, from the population, tothereby provide a population of CD14+ depleted cells to be expanded. Inone embodiment, the T regulatory cells, e.g., CD14+ cells, are removedby a method described herein.

In one embodiment, the method further comprises contacting thepopulation of immune effector cells with a nucleic acid encoding atelomerase subunit, e.g., hTERT. In an embodiment, the nucleic acid isDNA or RNA.

In another aspect, the disclosure features a reaction mixture comprisinga population of immune effector cells wherein a plurality of the cellsof the population in the reaction mixture comprise a nucleic acidmolecule, e.g., a nucleic acid molecule described herein, that comprisesa CAR encoding sequence, e.g., a CD19 CAR encoding sequence, e.g., asdescribed herein, and IL-7 and/or IL-15.

In one embodiment, a plurality of the cells of the population in thereaction mixture comprise a vector comprising a nucleic acid sequenceencoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR describedherein. In one embodiment, the vector is a vector described herein,e.g., a vector selected from the group consisting of a DNA, a RNA, aplasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

In one embodiment, the reaction mixture can further comprise an agentthat activates and/or expands to cells of the population, e.g., an agentthat stimulates a CD3/TCR complex associated signal and/or a ligand thatstimulates a costimulatory molecule on the surface of the cells, e.g.,as described herein. In one embodiment, the agent is a bead conjugatedwith anti-CD3 antibody, or a fragment thereof, and/or anti-CD28antibody, or a fragment thereof.

In another aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells) engineeredto express a CAR, the method comprising providing a population of immuneeffector cells (e.g., T cells), wherein a plurality of the immuneeffector cells comprise a nucleic acid encoding a CAR, e.g., a CARdescribed herein, e.g., a CD19 CAR described herein, and expanding thecells of the population in culture for 5 days, wherein the resultingcells are more potent, as measured by cell proliferation levels uponantigen stimulation, as compared to the same cells expanded in culturefor 9 days under the same culture conditions.

In one embodiment, the cells expanded for 5 days show at least a one,two, three or four fold increase in cells doublings upon antigenstimulation as compared to the same cells expanded in culture for 9 daysunder the same culture conditions. In one embodiment, the cells, e.g.,the cells expressing a CD19 CAR described herein, are expanded inculture for 5 days, and the resulting cells exhibit higherproinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels,as compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells expanded for 5days show at least a one, two, three, four, five, ten fold or moreincrease in pg/ml of proinflammatory cytokine production, e.g., IFN-γand/or GM-CSF levels, as compared to the same cells expanded in culturefor 9 days under the same culture conditions.

In another aspect, the disclosure features methods comprisingadministering to a subject a population of immune effector cells made bya method described herein and engineered to express a CAR, e.g. a CARdescribed herein, e.g., a CD19 CAR described herein.

In one embodiment, method provides an anti-tumor immunity in a subjecthaving cancer, e.g., a hematological cancer such as, e.g., CLL. In oneembodiment, the method is a method of treating a subject having cancer,e.g., a hematological cancer described herein, such as, e.g., a leukemia(e.g., CLL, ALL) or a lymphoma (e.g., MCL, HL). In one embodiment, thepopulation of cells are autologous to the subject administered thepopulation. In one embodiment, the population of cells is allogeneic tothe subject administered the population. In one embodiment, the subjectis a human.

In one embodiment, the disease associated with a tumor antigen, e.g., atumor antigen described herein, e.g., CD19, is selected from aproliferative disease such as a cancer or malignancy or a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia, or is a non-cancer related indication associated withexpression of a tumor antigen described herein. In one embodiment, thedisease is a cancer described herein, e.g., a cancer described herein asbeing associated with a target described herein. In one embodiment, thehematologic cancer is leukemia. In one embodiment, the cancer isselected from the group consisting of one or more acute leukemiasincluding but not limited to B-cell acute lymphoid leukemia (“BALL”),T-cell acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL);one or more chronic leukemias including but not limited to chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL);additional hematologic cancers or hematologic conditions including, butnot limited to B cell prolymphocytic leukemia, blastic plasmacytoiddendritic cell neoplasm, Burkitt's lymphoma, diffuse large B celllymphoma, follicular lymphoma, hairy cell leukemia, small cell- or alarge cell-follicular lymphoma, malignant lymphoproliferativeconditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma,multiple myeloma, myelodysplasia and myelodysplastic syndrome,non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendriticcell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia” whichare a diverse collection of hematological conditions united byineffective production (or dysplasia) of myeloid blood cells, and todisease associated with expression of a tumor antigen described hereininclude, but not limited to, atypical and/or non-classical cancers,malignancies, precancerous conditions or proliferative diseasesexpressing a tumor antigen as described herein; and any combinationthereof. In another embodiment, the disease associated with a tumorantigen described herein is a solid tumor, e.g., a solid tumor describedherein, e.g., prostatic, colorectal, pancreatic, cervical, gastric,ovarian, head, or lung cancer.

In one embodiment, the population of immune effector cells transducedwith a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g.,a CD19 CAR described herein, are expanded, e.g., by a method describedherein. In one embodiment, the cells are expanded for a period of 8 daysor less, e.g., 7, 6, 5, 4, or 3 days. In one embodiment, the cells,e.g., a CD19 CAR cell described herein, are expanded in culture for 5days, and the resulting cells are more potent than the same cellsexpanded in culture for 9 days under the same culture conditions, e.g.,as described herein. In one embodiment, the subject is administered 10⁴to 10⁶ immune effector cells per kg body weight of the subject. In oneembodiment, the subject receives an initial administration of apopulation of immune effector cells (e.g., an initial administration of10⁴ to 10⁶ immune effector cells per kg body weight of the subject,e.g., 10⁴ to 10⁵ immune effector cells per kg body weight of thesubject), a plurality of which comprise the nucleic acid encoding a CAR,e.g., a CAR described herein, e.g., a CD19 CAR described herein, and oneor more (e.g., 2, 3, 4, or 5) subsequent administrations of a populationof immune effector cells (e.g., one or more subsequent administration of10⁴ to 10⁶ immune effector cells per kg body weight of the subject,e.g., 10⁴ to 10⁵ immune effector cells per kg body weight of thesubject), a plurality of which comprise a nucleic acid encoding a CAR,e.g., a CAR described herein, e.g., a CD19 CAR described herein. In oneembodiment, the one or more subsequent administrations are administeredless than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2days after the previous administration, e.g., less than 4, 3, 2 daysafter the previous administration. In one embodiment, the subjectreceives a total of about 10⁶ immune effector cells per kg body weightof the subject over the course of at least three administrations of apopulation of immune effector cells, e.g., the subject receives aninitial dose of 1×10⁵ immune effector cells, a second administration of3×10⁵ immune effector cells, and a third administration of 6×10⁵ immuneeffector cells, and, e.g., each administration is administered less than4, 3, 2 days after the previous administration.

In one aspect, the disclosure features a population of autologous immuneeffector cells, a plurality of which are transfected or transduced witha vector comprising a nucleic acid molecule encoding a CAR, e.g., a CARdescribed herein, e.g., a CD19 CAR described herein, wherein thepopulation of cells contains less than 50%, 40%, 30%, 25%, 20%, 15%,10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 50%, 40%, 30%, 25%,20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CLL cells. Inone embodiment, the population of cells contains less than 15%, 10%, 5%,4%, 3%, 2%, 1% of CD25+ cells and less than 15%, 10%, 5%, 4%, 3%, 2%, 1%of tumor cells, e.g., CD25 expressing tumor cells, e.g., CLL cells. Inone embodiment, the population of cells contains less than 10%, 5%, 4%,3%, 2%, 1% of CD25+ cells and less than 10%, 5%, 4%, 3%, 2%, 1% of tumorcells, e.g., CD25 expressing tumor cells, e.g., CLL cells.

In another aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells). In anembodiment, the method comprises: providing a population of immuneeffector cells (e.g., T cells or NK cells), and contacting thepopulation of immune effector cells with a nucleic acid encoding a CAR,and a nucleic acid encoding a telomerase subunit, e.g., hTERT, underconditions that allow for CAR and telomerase expression.

In an embodiment, the nucleic acid encoding the telomerase subunit isRNA. In another embodiment, the nucleic acid encoding the telomerasesubunit is DNA. In an embodiment, the nucleic acid encoding thetelomerase subunit comprises a promoter capable of driving expression ofthe telomerase subunit.

In a related aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells) the methodcomprising: providing a population of immune effector cells (e.g., Tcells), contacting the population of immune effector cells with anucleic acid encoding a CAR and an RNA encoding a telomerase subunit,e.g., hTERT, under conditions that allow for CAR and telomeraseexpression.

In an embodiment, the nucleic acid encoding the CAR and the RNA encodingthe telomerase subunit are part of the same nucleic acid molecule. In anembodiment the nucleic acid encoding the CAR and the RNA encoding thetelomerase subunit are part of separate nucleic acid molecules.

In an embodiment, the method comprises contacting the population ofimmune effector cells with a nucleic acid encoding the CAR and the RNAencoding the telomerase subunit at substantially the same time. In anembodiment, the method comprises contacting the population of immuneeffector cells with a nucleic acid encoding the CAR before contactingthe population of immune effector cells with the RNA encoding thetelomerase subunit. In an embodiment, the method comprises contactingthe population of immune effector cells with a nucleic acid encoding theCAR after contacting the population of immune effector cells with theRNA encoding the telomerase subunit.

In an embodiment, the RNA encoding the telomerase subunit is mRNA. In anembodiment, the RNA encoding the telomerase subunit comprises a poly(A)tail. In an embodiment, the RNA encoding the telomerase subunitcomprises a 5′ cap structure.

In an embodiment, the method comprises transfecting the immune effectorcells with the RNA encoding the telomerase subunit. In an embodiment,the method comprises transducing the immune effector cells with the RNAencoding the telomerase subunit. In an embodiment, the method compriseselectroporating the immune effector cells with the RNA encoding thetelomerase subunit, under conditions that allow for CAR and telomeraseexpression.

In another aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells)comprising: providing a population of immune effector cells (e.g., Tcells or NK cells) that express a CAR and/or comprise a nucleic acidencoding a CAR; and contacting the population of immune effector cellswith a nucleic acid encoding a telomerase subunit, e.g., hTERT, underconditions that allow for hTERT expression.

In another aspect, the disclosure features a method of making apopulation of immune effector cells (e.g., T cells, NK cells),comprising: providing a population of immune effector cells (e.g., Tcells or NK cells) that express a nucleic acid encoding a telomerasesubunit, e.g., hTERT, and contacting the population of immune effectorcells with a nucleic acid encoding a CAR, under conditions that allowfor CAR expression.

In one aspect, this disclosure provides an immune effector cell (e.g., Tcell or NK cell) comprising: a nucleic acid encoding a CAR, e.g., a CARas described herein; and a nucleic acid encoding an exogenous telomerasesubunit, e.g., hTERT. In an embodiment, the nucleic acid encoding anexogenous telomerase subunit is RNA, e.g., mRNA.

In one aspect, this disclosure provides an immune effector cell (e.g., Tcell or NK cell) comprising: a CAR, e.g., a CAR as described herein; andan exogenous telomerase subunit, e.g., hTERT. In an embodiment, the celldoes not comprise DNA, e.g., exogenous DNA, e.g., a vector, encoding theexogenous telomerase subunit. For instance, the cell may have beencontacted with mRNA encoding the exogenous telomerase subunit.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references (e.g., sequencedatabase reference numbers) mentioned herein are incorporated byreference in their entirety. For example, all GenBank, Unigene, andEntrez sequences referred to herein, e.g., in any Table herein, areincorporated by reference. Unless otherwise specified, the sequenceaccession numbers specified herein, including in any Table herein, referto the database entries current as of Dec. 29, 2014. When one gene orprotein references a plurality of sequence accession numbers, all of thesequence variants are encompassed.

In addition, the materials, methods, and examples are illustrative onlyand not intended to be limiting. Headings, sub-headings or numbered orlettered elements, e.g., (a), (b), (i) etc., are presented merely forease of reading. The use of headings or numbered or lettered elements inthis document does not require the steps or elements be performed inalphabetical order or that the steps or elements are necessarilydiscrete from one another. Other features, objects, and advantages ofthe invention will be apparent from the description and drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H show the differential effects of γ_(c) cytokines and IL-18on CAR-T cell accumulation. FIG. 1A is a schematic diagram of C4-27z CARvector. FIG. 1B is two representative FACS histogram plots of CARexpression on CD4+ and CD8+ T cells 48 hours after lentiviraltransduction. FIG. 1C is a graph showing the overall accumulation ofCAR-T cells in response to various cytokines exposure. T cells weretransduced and exposed to various exogenous cytokines with finalconcentrations of 10 ng/mL from the next day (day 0). The numbers ofCAR-T cells were calculated based on the number of T cells and thepercentages of CAR expression. The curves are representative of 6donors. *P<0.05, ***P<0.001. NC, no cytokine. FIG. 1D is a graph showingCAR expression by T cells 15 days after lentiviral transduction. The bargraph depicts CAR expression levels (±SEM, n=6) on the surface of CD3+,CD4+ and CD8+ T cells, with the expression of CAR in NC group normalizedas 1. *P<0.05 versus IL-2 group; N.S., no statistical difference. FIG.1E is a histogram showing the proliferation of T cells in response tovarious cytokines. On day 7 after lentivirus transduction, T cells in NCgroup were labeled with CFSE (2.5 μM), and then exposed to variouscytokines. Seven days later, T cells were analyzed for CFSE dilution byflow cytometry. FIG. 1F is a graph showing the viability of T cells 15days after lentiviral transduction. T cells from various cytokine groupsare stained with Annexin V and 7-AAD, and then analyzed for theproportions of viable cells (both Annexin V and 7-AAD negative).*P<0.05, **P<0.01 versus IL-2 group (n=6). FIGS. 1G and 1H show Bcl-xLexpression by CAR-T cells. On day 15 days after lentiviral transduction,CAR-T cells are assessed for expression of Bcl-2 protein by flowcytometry. FIG. 1G is a representative FACS histogram plot of Bcl-xLexpression in various cytokine groups. FIG. 1H is a graph depictingBcl-xL expression (±SEM) in CD4+ and CD8+ CAR-T cells of 6 donors.*P<0.01 versus IL-2 group.

FIGS. 2A-2I shows the memory T cell subsets of CAR-T cells. FIG. 2A arerepresentative FACS plots showing the gating strategy of T cell subsetsanalysis. The T cells are split into four subsets based on CD45RA andCD62L expression, then the expression of CCR7, CD27, CD28 and CD95 arefurther evaluated for every subset. The CD95 expression is significantlyupregulated upon lentiviral transduction. FIG. 2B shows CD95 expressionin CD45RA+CD62L+ subpopulation of T cells before transduction and CAR-Tcells 15 days after transduction. FIGS. 2C and 2D are graphs showing theincrease of memory stem T cell (Tscm) proportions in CD4+(FIG. 2C) andCD8+ T cells (FIG. 2D) after lentiviral transduction. Tscm are definedas CD45RA+CD62L+CD95+CCR7+ T cell subsets. FIG. 2E is a graph showingthe correlation between the amount of naïve T (Tn, defined asCD45RA+CD62L+CD95− subpopulation) in T cells pre-transduction and theproportion of Tscm in CAR-T cells after transduction (n=6). Left barsrepresents the percentages of Tn in CD4+ and CD8+ T cells beforetransduction and right bars represents the percentages of Tscm in CD4+and CD8+ CAR-T cells. *P<0.05, **P<0.01. FIGS. 2F, 2G, and 2H are graphsshowing the distribution of subsets of CD4+ and CD8+ CAR-T cells. The Tsubsets in CD4+ and CD8+ CAR-T cells are defined based on the CD95,CD45RA and CD62L expression. The proportions of Tscm are compared amongvarious cytokine groups, *P<0.05, **P<0.01, versus IL-2 group (n=6). (F)Self-renew and differentiation of different subsets of CAR-T cells.FACS-sorted CAR+Tscm, Tcm, Tem and Temra cells are cultured exposed toIL-2 (10 ng/mL) for 3 days, then analyzed the phenotypes based on CD45RAand CD62L expression (n=3). FIG. 2I is a histogram plot showing theproliferation of various subsets of CAR-T cells in response to IL-2.FACS-sorted CAR+Tscm, Tcm, Tem and Temra cells were labeled with CFSE(2.5 μM), and then cultured exposed to IL-2 (10 ng/mL) for 3 days. Threedays later, T cells were analyzed for CFSE dilution.

FIGS. 3A-3B: shows the correlation between CD45 RA expression and CFSEintensity. FIG. 3A demonstrates that CD45 RAexpression is inverselycorrelated with CFSE intensity. FIG. 3B shows that for all cytokinegroups (IL-2, IL-7, IL-15, IL-18 and IL-21), CD45RA+ T cells exhibitedmuch lower CFSE levels than CD45RA dim and negative T cells indicatingthat CD45RA+ T cells had stronger proliferation activity than CD45RA− Tcells.

FIGS. 4A-4B show the phenotypes of CAR-T cells resulting from exposureto different cytokines. FIG. 4A is representative FACS dot-plots showingthe expression of CD45RA, CD62L, CCR7, CD27 and CD28 on CAR- and CAR+ Tcells exposed to IL-2 for 14 days after lentiviral transduction. FIG. 4Bis a series of graphs showing the quantitation of CD45RA, CD62L, CCR7,CD27, CD28 and IL7Ra expression on the surface of CAR-T cells inindicated cytokine groups. The histograms represent mean value±SEM ofexpression levels from 6 independent donors. *P<0.05, **P<0.01 versusIL-2 group.

FIGS. 5A-5I show the Functional analysis of CAR-T cells exposed todifferent cytokines. FIG. 5A is representative FACS plots show thestaining of intracellular IFN-γ, TNF-α and IL-2 in CAR-T cells. FIGS.5B, 5C, and 5D are quantitative plots showing the percentages ofcytokine-producing CAR-T cells in various cytokine groups (n=6) forproduction of IFNγ (FIG. 5B), TNF-α (FIG. 5C) and IL-2 (FIG. 5D).Lentiviral transduced T cells are exposed to indicated cytokines for 14days, and then co-cultured with SKOV3 cells for 5 hours before harvestedfor flow cytometry analysis. FIG. 5E is a series of pie charts depictingthe proportion of cells producing different numbers of cytokines (IFN-γ,TNF-α and IL-2) after SKOV3 stimulation. *P<0.05. FIG. 5F arerepresentative FACS plots showing the expression of perforin andgranzyme B (GranzB) in CAR-T cells. FIGS. 5G and 5H are quantitativeplots showing the percentages of perforin (FIG. 5G) and Granzyme Bexpression (FIG. 5H) in CAR-T cells in various cytokine groups (n=6).Lentiviral transduced T cells are exposed to indicated cytokines for 14days, and then co-cultured with SKOV3 cells for 5 hours before harvestedfor flow cytometry analysis. FIG. 5I is a graph showing the antigenspecific cytotoxic activity of CAR-T cells. Fourteen days afterindicated cytokine exposure, the CAR-T cells were assessed for cytolyticability by using a luciferase-based assay after 18-hour coculture withSKOV3 at the indicated E/T ratios. Untransduced T cells (UNT) served asnegative effector controls. Data shown are mean value±SEM of sixindependent cytolytic assays.

FIG. 6A-6C: shows the phenotype and function of the CAR-T cellsdescribed above in FIGS. 5A-5I. FIGS. 6A and 6B show that CD62L+CAR-Tcells (Tscm and Tcm) exhibited less cytokine production activity (FIGS.6A and 6B) and weaker cytolytic capacity (FIG. 6C) when compared withCD62L− CAR-T cells (Tem and Temra).

FIGS. 7A-7E show the expansion and phenotype of CAR-T cells exposed toantigen challenge. FIG. 7A is two graphs showing the overallaccumulation and viability of CAR-T cells in the exposure to antigen andindicated cytokines. The T cells exposed to IL-2 are harvested on day15, and then co-cultured with SKOV3 at E/T ratios of 5:1 and indicatedcytokines (10 ng/mL) for 7 days, with the supplement of SKOV3 cells onthe first and fourth day (The same protocol in FIGS. 7C, 7D, and 7E).The expansion folds are mean value±SEM. T cells are stained with AnnexinV and 7-AAD, and then analyzed for the proportions of viable cells atthe same day. *P<0.05 versus IL-2 group. FIG. 7B depicts two graphsshowing the overall accumulation and viability of CAR-T previouslyexposed to indicated cytokines upon antigen challenge. The T cellsexposed to indicated cytokines are harvested on day 15, and thenco-cultured with SKOV3 at E/T ratios of 5:1 for 7 days. The expansionsof CAR-T cells are calculated and the viability of T cells are evaluatedon the seventh day. FIG. 7C is a graph showing CAR expression by T cellsafter 7-day's coculture with SKOV3 and indicated cytokines. *P<0.05versus IL-2. FIG. 7D is two graphs showing the quantitation of CD27 andCD28 expression on CAR-T cells after 7-day's coculture with SKOV3 andindicated cytokines. *P<0.05 versus IL-2. FIG. 7E is two graphs showingthe distribution of memory T subsets of CD4+ and CD8+ CAR-T cells invarious cytokine groups. N.S., no statistical difference.

FIGS. 8A-8G show the antitumor activity of various CAR-T cells withprevious cytokine exposure. FIG. 8A is an in vivo experiment scheme.FIG. 8B Tumor growth curves of mice treated with various cytokineexposed C4-27z CAR-T cells, anti-CD19-27z CAR-T cells and untransduced Tcells. The data are presented as mean value±SEM. The arrow indicates thetime of T cell infusion. FIG. 8C is bioluminescence images showfLuc+SKOV3 tumors in NSG mice immediately before (day 38), two weeks(day53) and five weeks (day 74) after first intravenous injection ofCAR-T cells. FIG. 8D is a graph showing the quantitation of circulatinghuman CD4+ and CD8+ T cell counts in mice peripheral blood 15 days afterthe first dose of CAR-T cell infusion. FIG. 8E is a graph showing thequantitation of CAR expression on circulating human CD4+ and CD8+ Tcells in mice blood. FIG. 8F is a graph showing the distribution ofT-cell subsets of circulating human T cells in mice blood based onCD45RA and CD62L staining. FIG. 8G is a graph showing the quantitationof CD27 and CD28 expression on circulating human CD4+ and CD8+ T cellsin mice blood.

FIGS. 9A and 9B show FAC plots. FIG. 9A is a series of FACs plotsshowing the distribution of CD45, CD3, and CD25 expression in cells fromapheresis of a CLL patient. FIG. 9B is a series of FACS plots (top)showing the CD3 and CD19 populations and histograms (bottom) showingCD14 expression of cells from apheresis, cells selected withanti-CD3/CD28, cells depleted for CD25, and the CD25 enriched cells.

FIG. 10 is two FACs plots comparing the distribution of CD4+ and CD8+ Tcell populations after CD3/CD28 selection or CD25 depletion.

FIGS. 11A-11C show the comparison of proliferation capacity betweenCD3/CD28 selected cells and CD25 depleted cells. FIG. 11A is a graphshowing the total cell number at the indicated days in culture. FIG. 11Bis a graph showing the quantified population doublings at each indicatedday in culture. FIG. 11C shows the percentage of viable cells at theindicated days in culture.

FIGS. 12A and 12B show the effect of CD25 depletion on lentiviraltransduction of CAR19. FIG. 12A is a series of FACS plots showing theefficiency of CD25 depletion. FIG. 12B is a series of FACS plots showingthe CAR19 expression of untransduced cells, CD3 selected cells, and theCD25 depleted cells.

FIG. 13 is a series of FACS plots showing the distribution of CD3, CD19,and CD25 expression in cells from PBMCs from a patient before CD25depletion or culture with cytokine supplement.

FIG. 14 is a series of FACs plots showing the distribution of CD3 andCD19 in unmanipulated PBMCs and CD25-depleted PBMCs after culture withthe indicated cytokine supplements, IL-7, IL-15, or IL-7 and IL-15.

FIG. 15 is a graph showing the total number of cells after 10 days ofculture with the indicated cytokine supplements.

FIG. 16 is a graph showing the percentage of green fluorescent protein(GFP) signaling as an indication of lentiviral transfection levels fordonor cells transfected on two days, the day of (day 0) and the dayafter (day 1) the cells were obtained by apheresis (day 0+1); donorcells transfected once, the day of obtaining the cells by apheresis (day0); donor cells transfected once, the day after the cells were obtainedby apheresis (day 1); donor cells transfected once, two day after thecells were obtained by apheresis (day 2); and donor cells transfectedonce, the day after the cells were obtained by apheresis (day 3).

FIGS. 17A-17B comprise graphs showing expansion profile in populationdoublings (FIG. 17A) and mean size (fL)(FIG. 17B) of PBMCs that havebeen stimulated with anti-CD3 and CD28 beads, and left eitherunmanipulated (UTD) or transduced with a CD19 CAR (CD19.BBz), de-beaded,and then harvested at Day 5 and D9.

FIG. 18 comprises graphs depicting cytotoxicity as a percent lysis ofCD19 expressing K562 cells treated with PMBCs that have been stimulatedwith anti-CD3 and CD28 beads, and left either unmanipulated (UTD) ortransduced with a CD19 CAR (CD19.BBz), de-beaded, and then harvested atDay 5 and D9.

FIG. 19 comprises graphs depicting proliferation of PBMCs stimulatedwith anti-CD3 and CD28 beads (3×28 beads), wild type K562 cells, CD19expressing K562 cells, ALL cells (Nalm6) or CLL cells (PI14). The PBMCshave been left either unmanipulated (UTD) or transduced with a CD19 CAR(CART19), de-beaded, and then harvested at Day 5 and D9.

FIG. 20 is a schematic of an exemplary manufacturing scheme.

FIG. 21 is a schematic of an exemplary manufacturing scheme.

FIG. 22 comprises graphs depicting the level of cell proliferation oftwo different manufacturing batches of donor cells transfected with theCTL019 CAR, Patient 15 (left panels) and Patient 21 (right panels),expanded over a period of 0 to 9 days.

FIG. 23 comprises graphs showing proinflammatory cytokine production,IFN-γ, GM-CSF, TNF-α and IL-4 of two different manufacturing batches ofdonor cells transfected with either CTL019 CAR, namely Patient 15 cells,or an ss1-mesoCAR, namely Patient 21 cells, and expanded over a periodof 0 to 9 days after apheresis.

FIG. 24 comprises graphs depicting production levels IFN-γ, TNF-α, IL-6,IL-8, IL-2, IL-1β, GM-CSF and IL-4 in donor cells stimulated withanti-CAR19-idiotype antibody beads or control beads, transfected withCTL019 CAR and expanded for 5 to 9 days. No cytokine or low cytokinelevels (<200 pg/ml) were detected with the control beads.

FIG. 25 is a graph depicting cell killing based upon total lysates usinga luciferase assay of Nalm6 (ALL) cells of PBMCs left eitherunmanipulated (UTD) or transduced with a CD19 CAR. (CART19), de-beaded,and then harvested at Day 5 and D9. Various ratios of PMBCs to Nalm6cells (effector (E):Target (T)) were cultured. As shown CART19 cellsharvested at day 5 possess a better killing capacity.

FIG. 26 is a graph depicting long term in vivo killing capacity of PBMCsleft either unmanipulated (UTD) or transduced with a CD19 CAR (CART19),de-beaded, and then harvested at Day 5 and D9. The PBMCs were introducedinto non-obese diabetic/severe combined immunodeficiency mice inoculatedwith Nalm6 cells.

FIGS. 27A-27D indicate that bead removal at early timepoints does notinduce cell loss. FIG. 27A is a Coulter analysis illustrating mean Tcell volume and concentration before and after removing αCD3/αCD28coated magnetic beads via magnet on day 3. Cells were then frozen afterbead removal. FIG. 27B shows that cell number was evaluated before andafter removal of magnetic beads. The concentration was similar. FIG. 27Cis an overlay showing the mean cell volume of same cells before andafter thaw. Cells retain their volume during the freeze thaw process.Results are representative of at least 15 different experiments. FIG.27D shows a representative Coulter analysis of T cell products beforeand after αCD3/αCD28 coated magnetic bead removal demonstrating theefficiency of process.

FIGS. 28A-28B shows that T cells that are activated, transduced with aCD19-specific CAR bearing BBz signaling and harvested from T cellscultures at time points as early as day 3 following activation havepotent, antigen-specific cytotoxic activity in vitro. FIG. 28A, upperpanel, four-hour killing assay using K562-WT cells. FIG. 28B, lowerpanel, four-hour killing assay using K562-19 cells. FIG. 28B upperpanels, transduction efficiency using IL7/IL15 (left) or IL2 (right).FIG. 28B center panel, population doublings using IL7/IL15 compared toIL2. FIG. 28B lower panel, volume using IL7/IL15 compared to IL2.

FIGS. 29A-29C show that day 3 and day 9 4-1BBζ CART cells producesimilar patterns of cytokines in response to K562-CD19. Day 3 and day 9CART 19 cells, previously cultured in medium with IL-2 or IL-7/15, wereincubated with either K562-CD19 (FIGS. 29A and 29B), medium alone for 24h (FIG. 29C). “No antigen” in FIG. 29A and FIG. 29B indicates cellsstimulated with wild-type K562 cells without CD19. Cytokines weremeasured in culture supernatants by cytokine bead array (Luminex). FIG.29C shows representative cytokine production in CART 19 T cells 24 hafter thaw. The cytokine levels are minimal.

FIGS. 30A-30B show a progressive transition towards a moredifferentiated phenotype with an increasing proportion of Tem and Tcmcells.

FIG. 31A-31D. FIG. 31A shows the experimental setup for testing thepotency of CART cells produced under different conditions. FIG. 31Bshows the potency of IL2-treated CART cells in slowing Nalm6 tumorgrowth in mice. D3, D5, and D9 indicate CART cells that were harvestedon day 3, day 5, or day 9. 3e6 and 0.5e6 indicate the number of CAR+cells administered. Proportions of CAR+ cells are shown in FIG. 31C.FIG. 31D compares the potency of IL2-treated and IL7/IL15-treated CARTcells in slowing Nalm6 tumor growth in mice.

FIG. 32. In vitro expansion of CAR+ T cells in media contained IL-2 andIL-7/15, following antigen stimulation. Left panel, PBMCs from healthyhuman donors were stimulated with αCD3/αCD28 coated Dynal beads on day0, and lenti-virally transduced with 19-BK on day 1. These cells wereexpanded for 9 days in medium supplemented either with IL-2 or IL-7/15.T cells were counted by flow cytometry using bead-based counting everyother day. Right panel, Mean T cell volume (fl) over the course ofexpansion was monitored. Data are representative of at least sixindependent donors.

FIG. 33. Day 3 and day 9 CART19 cells cultured in medium with eitherIL-2 or IL-7/15 proliferate in response to K562-CD19, and not to K562-WTor medium alone. T cells were labeled with CFSE and co-cultured witheither K562-CD19, wild-type K562 or medium alone as a negative control,for 120 hours. The number of proliferating T cells was significantlyhigher in response to K562-CD19 as compared to wild-type K562 or mediumalone and was comparable between IL-7/15 and IL-2 groups. Results arerepresentative of two different donors.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

The term “a” and “an” refers to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of±20% or in some instances ±10%, or in some instances ±5%, or in someinstances ±1%, or in some instances ±0.1% from the specified value, assuch variations are appropriate to perform the disclosed methods.

The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers toa set of polypeptides, typically two in the simplest embodiments, whichwhen in an immune effector cell, provides the cell with specificity fora target cell, typically a cancer cell, and with intracellular signalgeneration. In some embodiments, a CAR comprises at least anextracellular antigen binding domain, a transmembrane domain and acytoplasmic signaling domain (also referred to herein as “anintracellular signaling domain”) comprising a functional signalingdomain derived from a stimulatory molecule and/or costimulatory moleculeas defined below. In some embodiments, the set of polypeptides are inthe same polypeptide chain (e.g., comprise a chimeric fusion protein).In some embodiments, the set of polypeptides are not contiguous witheach other, e.g., are in different polypeptide chains. In someembodiments, the set of polypeptides include a dimerization switch that,upon the presence of a dimerization molecule, can couple thepolypeptides to one another, e.g., can couple an antigen binding domainto an intracellular signaling domain. In one embodiment, the stimulatorymolecule of the CAR is the zeta chain associated with the T cellreceptor complex. In one aspect, the cytoplasmic signaling domaincomprises a primary signaling domain (e.g., a primary signaling domainof CD3-zeta). In one embodiment, the cytoplasmic signaling domainfurther comprises one or more functional signaling domains of at leastone costimulatory molecule as defined below. In one embodiment, thecostimulatory molecule is a costimulatory molecule described herein,e.g., 4-1BB (i.e., CD137), CD27, ICOS, and/or CD28. In one embodiment,the CAR comprises a chimeric fusion protein comprising an extracellularantigen binding domain, a transmembrane domain and an intracellularsignaling domain comprising a functional signaling domain of astimulatory molecule. In one embodiment, the CAR comprises a chimericfusion protein comprising an extracellular antigen binding domain, atransmembrane domain and an intracellular signaling domain comprising afunctional signaling domain of a co-stimulatory molecule and afunctional signaling domain of a stimulatory molecule. In oneembodiment, the CAR comprises a chimeric fusion protein comprising anextracellular antigen binding domain, a transmembrane domain and anintracellular signaling domain comprising two functional signalingdomains of one or more co-stimulatory molecule(s) and a functionalsignaling domain of a stimulatory molecule. In one embodiment, the CARcomprises a chimeric fusion protein comprising an extracellular antigenbinding domain, a transmembrane domain and an intracellular signalingdomain comprising at least two functional signaling domains of one ormore co-stimulatory molecule(s) and a functional signaling domain of astimulatory molecule. In one embodiment, the CAR comprises an optionalleader sequence at the amino-terminus (N-terminus) of the CAR fusionprotein. In one embodiment, the CAR further comprises a leader sequenceat the N-terminus of the extracellular antigen binding domain, whereinthe leader sequence is optionally cleaved from the antigen bindingdomain (e.g., a scFv) during cellular processing and localization of theCAR to the cellular membrane.

A CAR that comprises an antigen binding domain (e.g., a scFv, or TCR)that targets a specific tumor antigen X, such as those described herein,is also referred to as XCAR. For example, a CAR that comprises anantigen binding domain that targets CD19 is referred to as CD19CAR.

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers.

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules.

The term “antibody fragment” refers to at least one portion of anantibody, that retains the ability to specifically interact with (e.g.,by binding, steric hindrance, stabilizing/destabilizing, spatialdistribution) an epitope of an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, Fv fragments, scFvantibody fragments, disulfide-linked Fvs (sdFv), a Fd fragmentconsisting of the VH and CH1 domains, linear antibodies, single domainantibodies such as sdAb (either VL or VH), camelid VHH domains,multi-specific antibodies formed from antibody fragments such as abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, and an isolated CDR or other epitope bindingfragments of an antibody. An antigen binding fragment can also beincorporated into single domain antibodies, maxibodies, minibodies,nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology23:1126-1136, 2005). Antigen binding fragments can also be grafted intoscaffolds based on polypeptides such as a fibronectin type III (Fn3)(seeU.S. Pat. No. 6,703,199, which describes fibronectin polypeptideminibodies).

The term “scFv” refers to a fusion protein comprising at least oneantibody fragment comprising a variable region of a light chain and atleast one antibody fragment comprising a variable region of a heavychain, wherein the light and heavy chain variable regions arecontiguously linked, e.g., via a synthetic linker, e.g., a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the VL and VH variable regions in either order,e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise VL-linker-VH or may compriseVH-linker-VL.

The portion of a CAR comprising an antibody or antibody fragment thereofmay exist in a variety of forms where the antigen binding domain isexpressed as part of a contiguous polypeptide chain including, forexample, a single domain antibody fragment (sdAb), a single chainantibody (scFv) and a humanized antibody (Harlow et al., 1999, In: UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426). In oneembodiment, the antigen binding domain of a CAR comprises an antibodyfragment. In a further embodiment, the CAR comprises an antibodyfragment that comprises a scFv.

As used herein, the term “binding domain” or “antibody molecule” refersto a protein, e.g., an immunoglobulin chain or fragment thereof,comprising at least one immunoglobulin variable domain sequence. Theterm “binding domain” or “antibody molecule” encompasses antibodies andantibody fragments. In an embodiment, an antibody molecule is amultispecific antibody molecule, e.g., it comprises a plurality ofimmunoglobulin variable domain sequences, wherein a first immunoglobulinvariable domain sequence of the plurality has binding specificity for afirst epitope and a second immunoglobulin variable domain sequence ofthe plurality has binding specificity for a second epitope. In anembodiment, a multispecific antibody molecule is a bispecific antibodymolecule. A bispecific antibody has specificity for no more than twoantigens. A bispecific antibody molecule is characterized by a firstimmunoglobulin variable domain sequence which has binding specificityfor a first epitope and a second immunoglobulin variable domain sequencethat has binding specificity for a second epitope.

The portion of the CAR of the invention comprising an antibody orantibody fragment thereof may exist in a variety of forms where theantigen binding domain is expressed as part of a contiguous polypeptidechain including, for example, a single domain antibody fragment (sdAb),a single chain antibody (scFv), a humanized antibody, or bispecificantibody (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426). In one aspect, the antigen binding domain ofa CAR composition of the invention comprises an antibody fragment. In afurther aspect, the CAR comprises an antibody fragment that comprises ascFv.

The term “antibody heavy chain,” refers to the larger of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations, and which normally determines the class towhich the antibody belongs.

The term “antibody light chain,” refers to the smaller of the two typesof polypeptide chains present in antibody molecules in their naturallyoccurring conformations. Kappa (κ) and lambda (λ) light chains refer tothe two major antibody light chain isotypes.

The term “complementarity determining region” or “CDR,” as used herein,refers to the sequences of amino acids within antibody variable regionswhich confer antigen specificity and binding affinity. For example, ingeneral, there are three CDRs in each heavy chain variable region (e.g.,HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variableregion (LCDR1, LCDR2, and LCDR3). The precise amino acid sequenceboundaries of a given CDR can be determined using any of a number ofwell-known schemes, including those described by Kabat et al. (1991),“Sequences of Proteins of Immunological Interest,” 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (“Kabat” numberingscheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia”numbering scheme), or a combination thereof. Under the Kabat numberingscheme, in some embodiments, the CDR amino acid residues in the heavychain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2),and 95-102 (HCDR3); and the CDR amino acid residues in the light chainvariable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments,the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2),and 95-102 (HCDR3); and the CDR amino acid residues in the VL arenumbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combinedKabat and Chothia numbering scheme, in some embodiments, the CDRscorrespond to the amino acid residues that are part of a Kabat CDR, aChothia CDR, or both. For instance, in some embodiments, the CDRscorrespond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; andamino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in aVL, e.g., a mammalian VL, e.g., a human VL.

The term “recombinant antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage or yeast expression system. The term shouldalso be construed to mean an antibody which has been generated by thesynthesis of a DNA molecule encoding the antibody and which DNA moleculeexpresses an antibody protein, or an amino acid sequence specifying theantibody, wherein the DNA or amino acid sequence has been obtained usingrecombinant DNA or amino acid sequence technology which is available andwell known in the art.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.The skilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to encode polypeptides that elicit the desiredimmune response. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. It is readily apparentthat an antigen can be generated synthesized or can be derived from abiological sample, or might be macromolecule besides a polypeptide. Sucha biological sample can include, but is not limited to a tissue sample,a tumor sample, a cell or a fluid with other biological components.

The term “autologous” refers to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

The term “xenogeneic” refers to any material derived from an animal of adifferent species.

The term “cancer” refers to a disease characterized by the uncontrolledgrowth of aberrant cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. Examples ofvarious cancers are described herein and include but are not limited to,breast cancer, prostate cancer, ovarian cancer, cervical cancer, skincancer, pancreatic cancer, colorectal cancer, renal cancer, livercancer, brain cancer, lymphoma, leukemia, lung cancer and the like. Theterms “tumor” and “cancer” are used interchangeably herein, e.g., bothterms encompass solid and liquid, e.g., diffuse or circulating, tumors.As used herein, the term “cancer” or “tumor” includes premalignant, aswell as malignant cancers and tumors.

“Derived from” as that term is used herein, indicates a relationshipbetween a first and a second molecule. It generally refers to structuralsimilarity between the first molecule and a second molecule and does notconnote or include a process or source limitation on a first moleculethat is derived from a second molecule. For example, in the case of anintracellular signaling domain that is derived from a CD3zeta molecule,the intracellular signaling domain retains sufficient CD3zeta structuresuch that is has the required function, namely, the ability to generatea signal under the appropriate conditions. It does not connote orinclude a limitation to a particular process of producing theintracellular signaling domain, e.g., it does not mean that, to providethe intracellular signaling domain, one must start with a CD3zetasequence and delete unwanted sequence, or impose mutations, to arrive atthe intracellular signaling domain.

The phrase “disease associated with expression of a tumor antigen asdescribed herein” includes, but is not limited to, a disease associatedwith expression of a tumor antigen as described herein or conditionassociated with cells which express a tumor antigen as described hereinincluding, e.g., proliferative diseases such as a cancer or malignancyor a precancerous condition such as a myelodysplasia, a myelodysplasticsyndrome or a preleukemia; or a noncancer related indication associatedwith cells which express a tumor antigen as described herein. In oneembodiment, a cancer associated with expression of a tumor antigen asdescribed herein is a hematological cancer. In one embodiment, a cancerassociated with expression of a tumor antigen as described herein is asolid cancer. Further diseases associated with expression of a tumorantigen as described herein include, but not limited to, e.g., atypicaland/or non-classical cancers, malignancies, precancerous conditions orproliferative diseases associated with expression of a tumor antigen asdescribed herein. Non-cancer related indications associated withexpression of a tumor antigen as described herein include, but are notlimited to, e.g., autoimmune disease, (e.g., lupus), inflammatorydisorders (allergy and asthma) and transplantation. In some embodiments,the tumor antigen-expressing cells express, or at any time expressed,mRNA encoding the tumor antigen. In an embodiment, the tumorantigen-expressing cells produce the tumor antigen protein (e.g.,wild-type or mutant), and the tumor antigen protein may be present atnormal levels or reduced levels. In an embodiment, the tumorantigen-expressing cells produced detectable levels of a tumor antigenprotein at one point, and subsequently produced substantially nodetectable tumor antigen protein.

The phrase “disease associated with expression of CD19” includes, but isnot limited to, a disease associated with expression of CD19 orcondition associated with cells which express CD19 including, e.g.,proliferative diseases such as a cancer or malignancy or a precancerouscondition such as a myelodysplasia, a myelodysplastic syndrome or apreleukemia; or a noncancer related indication associated with cellswhich express CD19. In one aspect, a cancer associated with expressionof CD19 is a hematological cancer. In one aspect, the hematologicalcancer is a leukemia or a lymphoma. In one aspect, a cancer associatedwith expression of CD19 includes cancers and malignancies including, butnot limited to, e.g., one or more acute leukemias including but notlimited to, e.g., acute myeloid leukemia (AML), B-cell acute LymphoidLeukemia (BALL), T-cell acute Lymphoid Leukemia (TALL), acute lymphoidleukemia (ALL); one or more chronic leukemias including but not limitedto, e.g., chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia(CLL). Additional cancers or hematologic conditions associated withexpression of CD19 comprise, but are not limited to, e.g., B cellprolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm,Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma,Hairy cell leukemia, small cell- or a large cell-follicular lymphoma,malignant lymphoproliferative conditions, MALT lymphoma, mantle celllymphoma (MCL), Marginal zone lymphoma, multiple myeloma, myelodysplasiaand myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma,plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm,Waldenstrom macroglobulinemia, myeloproliferative neoplasm; ahistiocytic disorder (e.g., a mast cell disorder or a blasticplasmacytoid dendritic cell neoplasm); a mast cell disorder, e.g.,systemic mastocytosis or mast cell leukemia; B-cell prolymphocyticleukemia, plasma cell myeloma, and “preleukemia” which are a diversecollection of hematological conditions united by ineffective production(or dysplasia) of myeloid blood cells, and the like. Further diseasesassociated with expression of CD19 expression include, but not limitedto, e.g., atypical and/or non-classical cancers, malignancies,precancerous conditions or proliferative diseases associated withexpression of CD19. Non-cancer related indications associated withexpression of CD19 include, but are not limited to, e.g., autoimmunedisease, (e.g., lupus), inflammatory disorders (allergy and asthma) andtransplantation. In some embodiments, the tumor antigen-expressing cellsexpress, or at any time expressed, mRNA encoding the tumor antigen. Inan embodiment, the tumor antigen-expressing cells produce the tumorantigen protein (e.g., wild-type or mutant), and the tumor antigenprotein may be present at normal levels or reduced levels. In anembodiment, the tumor antigen-expressing cells produced detectablelevels of a tumor antigen protein at one point, and subsequentlyproduced substantially no detectable tumor antigen protein. In otherembodiments, the disease is a CD19-negative cancer, e.g., aCD19-negative relapsed cancer. In some embodiments, the tumor antigen(e.g., CD19)-expressing cell expresses, or at any time expressed, mRNAencoding the tumor antigen. In an embodiment, the tumor antigen (e.g.,CD19)-expressing cell produces the tumor antigen protein (e.g.,wild-type or mutant), and the tumor antigen protein may be present atnormal levels or reduced levels. In an embodiment, the tumor antigen(e.g., CD19)-expressing cell produced detectable levels of a tumorantigen protein at one point, and subsequently produced substantially nodetectable tumor antigen protein.

The phrase “disease associated with expression of a B-cell antigen”includes, but is not limited to, a disease associated with expression ofone or more of CD19, CD20, CD22 or ROR1, or a condition associated withcells which express, or at any time expressed, one or more of CD19,CD20, CD22 or ROR1, including, e.g., proliferative diseases such as acancer or malignancy or a precancerous condition such as amyelodysplasia, a myelodysplastic syndrome or a preleukemia; or anoncancer related indication associated with cells which express one ormore of CD19, CD20, CD22 or ROR1. For the avoidance of doubt, a diseaseassociated with expression of the B-cell antigen may include a conditionassociated with cells which do not presently express the B-cell antigen,e.g., because the antigen expression has been downregulated, e.g., dueto treatment with a molecule targeting the B-cell antigen, e.g., aB-cell targeting CAR, but which at one time expressed the antigen. Thephrase “disease associated with expression of a B-cell antigen” includesa disease associated with expression of CD19, as described herein. Inembodiments, the CAR-expressing cells are used to treat a diseaseassociated with a B-cell antigen. In embodiments, a CAR produced by amethod herein comprises an antigen binding domain that targets a B-cellantigen.

The term “relapse” as used herein refers to reappearance of a disease(e.g., cancer) after an initial period of responsiveness, e.g., afterprior treatment with a therapy, e.g., cancer therapy (e.g., completeresponse or partial response). The initial period of responsiveness mayinvolve the level of cancer cells falling below a certain threshold,e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance mayinvolve the level of cancer cells rising above a certain threshold,e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, e.g., inthe context of B-ALL, the reappearance may involve, e.g., a reappearanceof blasts in the blood, bone marrow (>5%), or any extramedullary site,after a complete response. A complete response, in this context, mayinvolve <5% BM blast. More generally, in an embodiment, a response(e.g., complete response or partial response) can involve the absence ofdetectable MRD (minimal residual disease). In an embodiment, the initialperiod of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; atleast 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months;or at least 1, 2, 3, 4, or 5 years.

“Refractory” as used herein refers to a disease, e.g., cancer, that doesnot respond to a treatment. In embodiments, a refractory cancer can beresistant to a treatment before or at the beginning of the treatment. Inother embodiments, the refractory cancer can become resistant during atreatment. A refractory cancer is also called a resistant cancer.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody or antibody fragment of the invention by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions are onesin which the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, one or more amino acid residues within a CAR describedherein can be replaced with other amino acid residues from the same sidechain family and the altered CAR can be tested using the functionalassays described herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with itscognate ligand (or tumor antigen in the case of a CAR) thereby mediatinga signal transduction event, such as, but not limited to, signaltransduction via the TCR/CD3 complex or signal transduction via theappropriate NK receptor or signaling domains of the CAR. Stimulation canmediate altered expression of certain molecules.

The term “stimulatory molecule,” refers to a molecule expressed by animmune cell (e.g., T cell, NK cell, B cell) that provides thecytoplasmic signaling sequence(s) that regulate activation of the immunecell in a stimulatory way for at least some aspect of the immune cellsignaling pathway. In one aspect, the signal is a primary signal that isinitiated by, for instance, binding of a TCR/CD3 complex with an MHCmolecule loaded with peptide, and which leads to mediation of a T cellresponse, including, but not limited to, proliferation, activation,differentiation, and the like. A primary cytoplasmic signaling sequence(also referred to as a “primary signaling domain”) that acts in astimulatory manner may contain a signaling motif which is known asimmunoreceptor tyrosine-based activation motif or ITAM. Examples of anITAM containing cytoplasmic signaling sequence that is of particular usein the invention includes, but is not limited to, those derived from CD3zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc EpsilonR1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.In a specific CAR of the invention, the intracellular signaling domainin any one or more CARS of the invention comprises an intracellularsignaling sequence, e.g., a primary signaling sequence of CD3-zeta. In aspecific CAR of the invention, the primary signaling sequence ofCD3-zeta is the sequence provided as SEQ ID NO:9 (mutant CD3 zeta), orthe equivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In a specific CAR of the invention, theprimary signaling sequence of CD3-zeta is the sequence as provided inSEQ ID NO:10 (wild-type human CD3 zeta), or the equivalent residues froma non-human species, e.g., mouse, rodent, monkey, ape and the like.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (MHC's) on its surface. T-cells mayrecognize these complexes using their T-cell receptors (TCRs). APCsprocess antigens and present them to T-cells.

An “intracellular signaling domain,” as the term is used herein, refersto an intracellular portion of a molecule. The intracellular signalingdomain can generate a signal that promotes an immune effector functionof the CAR containing cell, e.g., a CART cell. Examples of immuneeffector function, e.g., in a CART cell, include cytolytic activity andhelper activity, including the secretion of cytokines. In embodiments,the intracellular signaling domain is the portion of a protein whichtransduces the effector function signal and directs the cell to performa specialized function. While the entire intracellular signaling domaincan be employed, in many cases it is not necessary to use the entirechain. To the extent that a truncated portion of the intracellularsignaling domain is used, such truncated portion may be used in place ofthe intact chain as long as it transduces the effector function signal.The term intracellular signaling domain is thus meant to include anytruncated portion of the intracellular signaling domain sufficient totransduce the effector function signal.

In an embodiment, the intracellular signaling domain can comprise aprimary intracellular signaling domain. Exemplary primary intracellularsignaling domains include those derived from the molecules responsiblefor primary stimulation, or antigen dependent simulation. In anembodiment, the intracellular signaling domain can comprise acostimulatory intracellular domain. Exemplary costimulatoryintracellular signaling domains include those derived from moleculesresponsible for costimulatory signals, or antigen independentstimulation. For example, in the case of a CART, a primary intracellularsignaling domain can comprise a cytoplasmic sequence of a T cellreceptor, and a costimulatory intracellular signaling domain cancomprise cytoplasmic sequence from co-receptor or costimulatorymolecule.

A primary intracellular signaling domain can comprise a signaling motifwhich is known as an immunoreceptor tyrosine-based activation motif orITAM. Examples of ITAM containing primary cytoplasmic signalingsequences include, but are not limited to, those derived from CD3 zeta,FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, CD278 (“ICOS”), FcεRI, and CD66d, CD32, DAP10, and DAP12.

The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta”is defined as the protein provided as GenBank Acc. No. BAG36664.1, orthe equivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like, and a “zeta stimulatory domain” oralternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatorydomain” is defined as the amino acid residues from the cytoplasmicdomain of the zeta chain that are sufficient to functionally transmit aninitial signal necessary for T cell activation. In one aspect thecytoplasmic domain of zeta comprises residues 52 through 164 of GenBankAcc. No. BAG36664.1 or the equivalent residues from a non-human species,e.g., mouse, rodent, monkey, ape and the like, that are functionalorthologs thereof. In one aspect, the “zeta stimulatory domain” or a“CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO:9.In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatorydomain” is the sequence provided as SEQ ID NO:10.

The term “costimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a costimulatory ligand, therebymediating a costimulatory response by the T cell, such as, but notlimited to, proliferation. Costimulatory molecules are cell surfacemolecules other than antigen receptors or their ligands that arerequired for an efficient immune response. Costimulatory moleculesinclude, but are not limited to MHC class I molecule, TNF receptorproteins, Immunoglobulin-like proteins, cytokine receptors, integrins,signalling lymphocytic activation molecules (SLAM proteins), activatingNK cell receptors, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27,CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3,CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2,SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS,SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain refers to anintracellular portion of a costimulatory molecule. The intracellularsignaling domain can comprise the entire intracellular portion, or theentire native intracellular signaling domain, of the molecule from whichit is derived, or a functional fragment thereof.

The intracellular signaling domain can comprise the entire intracellularportion, or the entire native intracellular signaling domain, of themolecule from which it is derived, or a functional fragment thereof.

The term “4-1BB” refers to a member of the TNFR superfamily with anamino acid sequence provided as GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like; and a “4-1BB costimulatory domain” is definedas amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or theequivalent residues from a non-human species, e.g., mouse, rodent,monkey, ape and the like. In one aspect, the “4-1BB costimulatorydomain” is the sequence provided as SEQ ID NO:7 or the equivalentresidues from a non-human species, e.g., mouse, rodent, monkey, ape andthe like.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response. Examples of immune effector cells include Tcells, e.g., alpha/beta T cells and gamma/delta T cells, B cells,natural killer (NK) cells, natural killer T (NKT) cells, mast cells, andmyeloid-derived phagocytes.

“Immune effector function or immune effector response,” as that term isused herein, refers to function or response, e.g., of an immune effectorcell, that enhances or promotes an immune attack of a target cell. E.g.,an immune effector function or response refers a property of a T or NKcell that promotes killing or the inhibition of growth or proliferation,of a target cell. In the case of a T cell, primary stimulation andco-stimulation are examples of immune effector function or response.

The term “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene, cDNA, or RNA, encodes a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell orother biological system. Both the coding strand, the nucleotide sequenceof which is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or a RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

The term “transfer vector” refers to a composition of matter whichcomprises an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, including cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic, include but are not limited to, e.g., the LENTIVECTOR®gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies and antibody fragments thereofare human immunoglobulins (recipient antibody or antibody fragment) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, a humanizedantibody/antibody fragment can comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. These modifications can further refine and optimize antibodyor antibody fragment performance. In general, the humanized antibody orantibody fragment thereof will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or a significant portion of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody or antibody fragment canalso comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details, seeJones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody orantibody fragment, where the whole molecule is of human origin orconsists of an amino acid sequence identical to a human form of theantibody or immunoglobulin.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “parenteral” administration of an immunogenic compositionincludes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular(i.m.), or intrasternal injection, intratumoral, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNAthereof, and polymers thereof in either single- or double-stranded form.The term “nucleic acid” includes a gene, cDNA or an mRNA. In oneembodiment, the nucleic acid molecule is synthetic (e.g., chemicallysynthesized) or recombinant. Unless specifically limited, the termencompasses nucleic acids containing analogues or derivatives of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence.

The term “promoter/regulatory sequence” refers to a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter/regulatory sequence. In some instances, thissequence may be the core promoter sequence and in other instances, thissequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The terms “cancer associated antigen” or “tumor antigen” interchangeablyrefers to a molecule (typically protein, carbohydrate or lipid) that ispreferentially expressed on the surface of a cancer cell, eitherentirely or as a fragment (e.g., MHC/peptide), in comparison to a normalcell, and which is useful for the preferential targeting of apharmacological agent to the cancer cell. In some embodiments, a tumorantigen is a marker expressed by both normal cells and cancer cells,e.g., a lineage marker, e.g., CD19 on B cells. In certain aspects, thetumor antigens of the present invention are derived from, cancersincluding but not limited to primary or metastatic melanoma, thymoma,lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma,Hodgkin lymphoma, leukemias, uterine cancer, cervical cancer, bladdercancer, kidney cancer and adenocarcinomas such as breast cancer,prostate cancer, ovarian cancer, pancreatic cancer, and the like. Insome embodiments, a cancer-associated antigen is a cell surface moleculethat is overexpressed in a cancer cell in comparison to a normal cell,for instance, 1-fold over expression, 2-fold overexpression, 3-foldoverexpression or more in comparison to a normal cell. In someembodiments, a cancer-associated antigen is a cell surface molecule thatis inappropriately synthesized in the cancer cell, for instance, amolecule that contains deletions, additions or mutations in comparisonto the molecule expressed on a normal cell. In some embodiments, acancer-associated antigen will be expressed exclusively on the cellsurface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide),and not synthesized or expressed on the surface of a normal cell. Insome embodiments, the CARs of the present invention includes CARscomprising an antigen binding domain (e.g., antibody or antibodyfragment) that binds to a MHC presented peptide. Normally, peptidesderived from endogenous proteins fill the pockets of Majorhistocompatibility complex (MHC) class I molecules, and are recognizedby T cell receptors (TCRs) on CD8+ T lymphocytes. The MHC class Icomplexes are constitutively expressed by all nucleated cells. Incancer, virus-specific and/or tumor-specific peptide/MHC complexesrepresent a unique class of cell surface targets for immunotherapy.TCR-like antibodies targeting peptides derived from viral or tumorantigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2have been described (see, e.g., Sastry et al., J Virol. 201185(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Vermaet al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33;Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example,TCR-like antibody can be identified from screening a library, such as ahuman scFv phage displayed library.

The term “flexible polypeptide linker” or “linker” as used in thecontext of a scFv refers to a peptide linker that consists of aminoacids such as glycine and/or serine residues used alone or incombination, to link variable heavy and variable light chain regionstogether. In one embodiment, the flexible polypeptide linker is aGly/Ser linker and comprises the amino acid sequence(Gly-Gly-Gly-Ser)_(n) (SEQ ID NO: 15), where n is a positive integerequal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6,n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptidelinkers include, but are not limited to, (Gly₄ Ser)₄ (SEQ ID NO:27) or(Gly₄ Ser)₃ (SEQ ID NO:28). In another embodiment, the linkers includemultiple repeats of (Gly₂Ser), (GlySer) or (Gly₃Ser) (SEQ ID NO:29).Also included within the scope of the invention are linkers described inWO2012/138475, incorporated herein by reference).

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m⁷G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

As used herein, “in vitro transcribed RNA” refers to RNA, e.g., mRNA,that has been synthesized in vitro. Generally, the in vitro transcribedRNA is generated from an in vitro transcription vector. The in vitrotranscription vector comprises a template that is used to generate thein vitro transcribed RNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In some embodiments of a construct fortransient expression, the polyA is between 50 and 5000 (SEQ ID NO: 30),e.g., greater than 64, e.g., greater than 100, e.g., greater than 300 or400 poly(A) sequences can be modified chemically or enzymatically tomodulate mRNA functionality such as localization, stability orefficiency of translation.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

Apheresis is the process in which whole blood is removed from anindividual, separated into select components, and the remainder returnedto circulation. Generally, there are two methods for the separation ofblood components, centrifugal and non-centrifugal. Leukapheresis resultsin the active selection and removal of the patient's white blood cells.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a proliferative disorder, or the amelioration of one or moresymptoms (e.g., one or more discernible symptoms) of a proliferativedisorder resulting from the administration of one or more therapies(e.g., one or more therapeutic agents such as a CAR of the invention).In specific embodiments, the terms “treat”, “treatment” and “treating”refer to the amelioration of at least one measurable physical parameterof a proliferative disorder, such as growth of a tumor, not necessarilydiscernible by the patient. In other embodiments the terms “treat”,“treatment” and “treating”-refer to the inhibition of the progression ofa proliferative disorder, either physically by, e.g., stabilization of adiscernible symptom, physiologically by, e.g., stabilization of aphysical parameter, or both. In other embodiments the terms “treat”,“treatment” and “treating” refer to the reduction or stabilization oftumor size or cancerous cell count.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human).

The term, a “substantially purified” cell refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

In the context of the present invention, “tumor antigen” or“hyperproliferative disorder antigen” or “antigen associated with ahyperproliferative disorder” refers to antigens that are common tospecific hyperproliferative disorders. In certain embodiments, the tumorantigen is derived from a cancer including but not limited to primary ormetastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, livercancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterinecancer, cervical cancer, bladder cancer, kidney cancer andadenocarcinomas such as breast cancer, prostate cancer, ovarian cancer,pancreatic cancer, and the like.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “specifically binds,” refers to an antibody, or a ligand, whichrecognizes and binds with a cognate binding partner protein present in asample, but which antibody or ligand does not substantially recognize orbind other molecules in the sample.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity, includes somethingwith 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

Description

Provided herein are methods of manufacturing immune effector cells(e.g., T cells, NK cells) that can be engineered with a CAR, e.g., a CARdescribed herein, and reaction mixtures and compositions comprising suchcells.

In one aspect, the disclosure features an immune effector cell (e.g., Tcell, NK cell) engineered to express a CAR, wherein the engineeredimmune effector cell exhibits an antitumor property. An exemplaryantigen is a cancer associated antigen (i.e., tumor antigen) describedherein. In one aspect, a cell is transformed with the CAR and the CAR isexpressed on the cell surface. In some embodiments, the cell (e.g., Tcell, NK cell) is transduced with a viral vector encoding a CAR. In someembodiments, the viral vector is a retroviral vector. In someembodiments, the viral vector is a lentiviral vector. In some suchembodiments, the cell may stably express the CAR. In another embodiment,the cell (e.g., T cell, NK cell) is transfected with a nucleic acid,e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, thecell may transiently express the CAR.

Furthermore, the present invention provides CAR-expressing cell, e.g.,CART compositions and their use in medicaments or methods for treating,among other diseases, cancer or any malignancy or autoimmune diseasesinvolving cells or tissues which express a tumor antigen as describedherein.

In one aspect, the CAR of the invention can be used to eradicate anormal cell that express a tumor antigen as described herein, therebyapplicable for use as a cellular conditioning therapy prior to celltransplantation.

Sources of Immune Effector Cells

In embodiments, prior to expansion and genetic modification or othermodification, a source of cells, e.g., T cells or natural killer (NK)cells, can be obtained from a subject. Examples of subjects includehumans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenicspecies thereof. T cells can be obtained from a number of sources,including peripheral blood mononuclear cells, bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors.

In certain aspects of the present disclosure, immune effector cells,e.g., T cells, can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one aspect, cells from the circulatingblood of an individual are obtained by apheresis. The apheresis producttypically contains lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and platelets. In one aspect, the cells collected by apheresismay be washed to remove the plasma fraction and, optionally, to placethe cells in an appropriate buffer or media for subsequent processingsteps. In one embodiment, the cells are washed with phosphate bufferedsaline (PBS). In an alternative embodiment, the wash solution lackscalcium and may lack magnesium or may lack many if not all divalentcations.

Initial activation steps in the absence of calcium can lead to magnifiedactivation. As those of ordinary skill in the art would readilyappreciate a washing step may be accomplished by methods known to thosein the art, such as by using a semi-automated “flow-through” centrifuge(for example, the Cobe 2991 cell processor, the Baxter CytoMate, or theHaemonetics Cell Saver 5) according to the manufacturer's instructions.After washing, the cells may be resuspended in a variety ofbiocompatible buffers, such as, for example, Ca-free, Mg-free PBS,PlasmaLyte A, or other saline solution with or without buffer.Alternatively, the undesirable components of the apheresis sample may beremoved and the cells directly resuspended in culture media.

In one aspect, T cells are isolated from peripheral blood lymphocytes bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient or by counterflow centrifugalelutriation.

The methods described herein can include, e.g., selection of a specificsubpopulation of immune effector cells, e.g., T cells, that are a Tregulatory cell-depleted population, CD25+ depleted cells, using, e.g.,a negative selection technique, e.g., described herein. In someembodiments, the population of T regulatory-depleted cells contains lessthan 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removedfrom the population using an anti-CD25 antibody, or fragment thereof, ora CD25-binding ligand, e.g. IL-2. In one embodiment, the anti-CD25antibody, or fragment thereof, or CD25-binding ligand is conjugated to asubstrate, e.g., a bead, or is otherwise coated on a substrate, e.g., abead. In one embodiment, the anti-CD25 antibody, or fragment thereof, isconjugated to a substrate as described herein.

In one embodiment, the T regulatory cells, e.g., CD25+ T cells, areremoved from the population using CD25 depleting reagent from Miltenyi™.In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In oneembodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greaterthan 500 million cells/ml is used. In a further aspect, a concentrationof cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to bedepleted includes about 6×10⁹ CD25+ T cells. In other aspects, thepopulation of immune effector cells to be depleted include about 1×10⁹to 1×10¹⁰ CD25+ T cell, and any integer value in between. In oneembodiment, the resulting population T regulatory-depleted cells has2×10⁹ T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10⁹,5×10⁸, 1×10⁸, 5×10⁷, 1×10⁷, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, areremoved from the population using the CliniMAC system with a depletiontubing set, such as, e.g., tubing 162-01. In one embodiment, theCliniMAC system is run on a depletion setting such as, e.g.,DEPLETION2.1.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell productsignificantly reduces the risk of subject relapse. For example, methodsof depleting T_(REG) cells are known in the art. Methods of decreasingT_(REG) cells include, but are not limited to, cyclophosphamide,anti-GITR antibody (an anti-GITR antibody described herein),CD25-depletion, mTOR inhibitor, and combinations thereof.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

Without wishing to be bound by a particular theory, decreasing the levelof negative regulators of immune cells (e.g., decreasing the number ofunwanted immune cells, e.g., T_(REG) cells), in a subject prior toapheresis or during manufacturing of a CAR-expressing cell product canreduce the risk of a subject's relapse. In an embodiment, a subject ispre-treated with one or more therapies that reduce T_(REG) cells priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment. In an embodiment, methods of decreasing T_(REG) cellsinclude, but are not limited to, administration to the subject of one ormore of cyclophosphamide, anti-GITR antibody, CD25-depletion, or acombination thereof. In an embodiment, methods of decreasing T_(REG)cells include, but are not limited to, administration to the subject ofone or more of cyclophosphamide, anti-GITR antibody, CD25-depletion,mTOR inhibitor, or a combination thereof. Administration of one or moreof cyclophosphamide, anti-GITR antibody, CD25-depletion, or acombination thereof, can occur before, during or after an infusion ofthe CAR-expressing cell product. Administration of one or more ofcyclophosphamide, anti-GITR antibody, CD25-depletion, mTOR inhibitor, ora combination thereof, can occur before, during or after an infusion ofthe CAR-expressing cell product.

In some embodiments, the manufacturing methods comprise reducing thenumber of (e.g., depleting) T_(REG) cells prior to manufacturing of theCAR-expressing cell. For example, manufacturing methods comprisecontacting the sample, e.g., the apheresis sample, with an anti-GITRantibody and/or an anti-CD25 antibody (or fragment thereof, or aCD25-binding ligand), e.g., to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell)product.

In an embodiment, a subject is pre-treated with cyclophosphamide priorto collection of cells for CAR-expressing cell product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment (e.g., CTL019 treatment). In an embodiment, a subject ispre-treated with an anti-GITR antibody prior to collection of cells forCAR-expressing cell (e.g., T cell or NK cell) product manufacturing,thereby reducing the risk of subject relapse to CAR-expressing celltreatment.

In an embodiment, the CAR-expressing cell (e.g., T cell, NK cell)manufacturing process is modified to deplete T_(REG) cells prior tomanufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product(e.g., a CTL019 product). In an embodiment, CD25-depletion is used todeplete T_(REG) cells prior to manufacturing of the CAR-expressing cell(e.g., T cell, NK cell) product (e.g., a CTL019 product).

In one embodiment, the population of cells to be removed are neither theregulatory T cells or tumor cells, but cells that otherwise negativelyaffect the expansion and/or function of CART cells, e.g. cellsexpressing CD14, CD11b, CD33, CD15, or other markers expressed bypotentially immune suppressive cells. In one embodiment, such cells areenvisioned to be removed concurrently with regulatory T cells and/ortumor cells, or following said depletion, or in another order.

The methods described herein can include more than one selection step,e.g., more than one depletion step. Enrichment of a T cell population bynegative selection can be accomplished, e.g., with a combination ofantibodies directed to surface markers unique to the negatively selectedcells. One method is cell sorting and/or selection via negative magneticimmunoadherence or flow cytometry that uses a cocktail of monoclonalantibodies directed to cell surface markers present on the cellsnegatively selected. For example, to enrich for CD4+ cells by negativeselection, a monoclonal antibody cocktail can include antibodies toCD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from thepopulation which express a tumor antigen, e.g., a tumor antigen thatdoes not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 orCD11b, to thereby provide a population of T regulatory-depleted, e.g.,CD25+ depleted, and tumor antigen depleted cells that are suitable forexpression of a CAR, e.g., a CAR described herein. In one embodiment,tumor antigen expressing cells are removed simultaneously with the Tregulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, orfragment thereof, and an anti-tumor antigen antibody, or fragmentthereof, can be attached to the same substrate, e.g., bead, which can beused to remove the cells or an anti-CD25 antibody, or fragment thereof,or the anti-tumor antigen antibody, or fragment thereof, can be attachedto separate beads, a mixture of which can be used to remove the cells.In other embodiments, the removal of T regulatory cells, e.g., CD25+cells, and the removal of the tumor antigen expressing cells issequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from thepopulation which express a check point inhibitor, e.g., a check pointinhibitor described herein, e.g., one or more of PD1+ cells, LAG3+cells, and TIM3+ cells, to thereby provide a population of Tregulatory-depleted, e.g., CD25+ depleted cells, and check pointinhibitor depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells.Exemplary check point inhibitors include PD1, PD-L1, PD-L2, CTLA4, TIM3,CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GALS,adenosine, and TGFR (e.g., TGFRbeta), e.g., as described herein. In oneembodiment, check point inhibitor expressing cells are removedsimultaneously with the T regulatory, e.g., CD25+ cells. For example, ananti-CD25 antibody, or fragment thereof, and an anti-check pointinhibitor antibody, or fragment thereof, can be attached to the samebead which can be used to remove the cells, or an anti-CD25 antibody, orfragment thereof, and the anti-check point inhibitor antibody, orfragment thereof, can be attached to separate beads, a mixture of whichcan be used to remove the cells. In other embodiments, the removal of Tregulatory cells, e.g., CD25+ cells, and the removal of the check pointinhibitor expressing cells is sequential, and can occur, e.g., in eitherorder.

Methods described herein can include a positive selection step. Forexample, T cells can isolated by incubation with anti-CD3/anti-CD28(e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, fora time period sufficient for positive selection of the desired T cells.In one embodiment, the time period is about 30 minutes. In a furtherembodiment, the time period ranges from 30 minutes to 36 hours or longerand all integer values there between. In a further embodiment, the timeperiod is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment,the time period is 10 to 24 hours, e.g., 24 hours. Longer incubationtimes may be used to isolate T cells in any situation where there arefew T cells as compared to other cell types, such in isolating tumorinfiltrating lymphocytes (TIL) from tumor tissue or fromimmunocompromised individuals. Further, use of longer incubation timescan increase the efficiency of capture of CD8+ T cells. Thus, by simplyshortening or lengthening the time T cells are allowed to bind to theCD3/CD28 beads and/or by increasing or decreasing the ratio of beads toT cells (as described further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints.

In one embodiment, a T cell population can be selected that expressesone or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10,IL-13, granzyme B, and perforin, or other appropriate molecules, e.g.,other cytokines. Methods for screening for cell expression can bedetermined, e.g., by the methods described in PCT Publication No.: WO2013/126712.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain aspects, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (e.g., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one aspect, a concentrationof 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1billion cells/ml is used. In yet one aspect, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtheraspects, concentrations of 125 or 150 million cells/ml can be used.

Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (e.g., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8+ T cells that normally haveweaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations ofcells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4+ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8+ T cells in dilute concentrations. In one aspect, the concentrationof cells used is 5×10⁶/ml. In other aspects, the concentration used canbe from about 1×10⁵/ml to 1×10⁶/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varyinglengths of time at varying speeds at either 2-10° C. or at roomtemperature.

In one embodiment, a plurality of the immune effector cells of thepopulation do not express diaglycerol kinase (DGK), e.g., isDGK-deficient. In one embodiment, a plurality of the immune effectorcells of the population do not express Ikaros, e.g., isIkaros-deficient. In one embodiment, a plurality of the immune effectorcells of the population do not express DGK and Ikaros, e.g., is both DGKand Ikaros-deficient.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in immune effector cell therapy for any number of diseasesor conditions that would benefit from immune effector cell therapy, suchas those described herein. In one aspect a blood sample or an apheresisis taken from a generally healthy subject. In certain aspects, a bloodsample or an apheresis is taken from a generally healthy subject who isat risk of developing a disease, but who has not yet developed adisease, and the cells of interest are isolated and frozen for lateruse. In certain aspects, the T cells may be expanded, frozen, and usedat a later time. In certain aspects, samples are collected from apatient shortly after diagnosis of a particular disease as describedherein but prior to any treatments. In a further aspect, the cells areisolated from a blood sample or an apheresis from a subject prior to anynumber of relevant treatment modalities, including but not limited totreatment with agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained froma patient directly following treatment that leaves the subject withfunctional T cells. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated within thecontext of the present invention to collect blood cells, including Tcells, dendritic cells, or other cells of the hematopoietic lineage,during this recovery phase. Further, in certain aspects, mobilization(for example, mobilization with GM-CSF) and conditioning regimens can beused to create a condition in a subject wherein repopulation,recirculation, regeneration, and/or expansion of particular cell typesis favored, especially during a defined window of time followingtherapy. Illustrative cell types include T cells, B cells, dendriticcells, and other cells of the immune system.

In one embodiment, the immune effector cells expressing a CAR molecule,e.g., a CAR molecule described herein, are obtained from a subject thathas received a low, immune enhancing dose of an mTOR inhibitor. In anembodiment, the population of immune effector cells, e.g., T cells, tobe engineered to express a CAR, are harvested after a sufficient time,or after sufficient dosing of the low, immune enhancing, dose of an mTORinhibitor, such that the level of PD1 negative immune effector cells,e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g.,T cells/PD1 positive immune effector cells, e.g., T cells, in thesubject or harvested from the subject has been, at least transiently,increased.

In other embodiments, population of immune effector cells, e.g., Tcells, which have, or will be engineered to express a CAR, can betreated ex vivo by contact with an amount of an mTOR inhibitor thatincreases the number of PD1 negative immune effector cells, e.g., Tcells or increases the ratio of PD1 negative immune effector cells,e.g., T cells/PD1 positive immune effector cells, e.g., T cells.

It is recognized that the methods of the application can utilize culturemedia conditions comprising 5% or less, for example 2%, human AB serum,and employ known culture media conditions and compositions, for examplethose described in Smith et al., “Ex vivo expansion of human T cells foradoptive immunotherapy using the novel Xeno-free CTS Immune Cell SerumReplacement” Clinical & Translational Immunology (2015) 4, e31;doi:10.1038/cti.2014.31.

In one embodiment, the methods of the application can utilize culturemedia conditions comprising serum-free medium. In one embodiment, theserum free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcelltechnologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline(Sigma), Xvivo15 (Lonza), PrimeXV (Irvine Scientific), or StemXVivo(RandD systems). The serum-free medium can be supplemented with a serumsubstitute such as ICSR (immune cell serum replacement) from LifeTech.The level of serum substitute (e.g., ICSR) can be, e.g., up to 5%, e.g.,about 1%, 2%, 3%, 4%, or 5%.

In one embodiment, a T cell population is diaglycerol kinase(DGK)-deficient. DGK-deficient cells include cells that do not expressDGK RNA or protein, or have reduced or inhibited DGK activity.DGK-deficient cells can be generated by genetic approaches, e.g.,administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, toreduce or prevent DGK expression. Alternatively, DGK-deficient cells canbe generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient.Ikaros-deficient cells include cells that do not express Ikaros RNA orprotein, or have reduced or inhibited Ikaros activity, Ikaros-deficientcells can be generated by genetic approaches, e.g., administeringRNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or preventIkaros expression. Alternatively, Ikaros-deficient cells can begenerated by treatment with Ikaros inhibitors, e.g., lenalidomide.

In embodiments, a T cell population is DGK-deficient andIkaros-deficient, e.g., does not express DGK and Ikaros, or has reducedor inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficientcells can be generated by any of the methods described herein.

In an embodiment, the NK cells are obtained from the subject. In anotherembodiment, the NK cells are an NK cell line, e.g., NK-92 cell line(Conkwest).

Allogeneic CAR

In embodiments described herein, the immune effector cell can be anallogeneic immune effector cell, e.g., T cell or NK cell. For example,the cell can be an allogeneic T cell, e.g., an allogeneic T cell lackingexpression of a functional T cell receptor (TCR) and/or human leukocyteantigen (HLA), e.g., HLA class I and/or HLA class II.

A T cell lacking a functional TCR can be, e.g., engineered such that itdoes not express any functional TCR on its surface, engineered such thatit does not express one or more subunits that comprise a functional TCR(e.g., engineered such that it does not express (or exhibits reducedexpression) of TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon,and/or TCR zeta) or engineered such that it produces very littlefunctional TCR on its surface. Alternatively, the T cell can express asubstantially impaired TCR, e.g., by expression of mutated or truncatedforms of one or more of the subunits of the TCR. The term “substantiallyimpaired TCR” means that this TCR will not elicit an adverse immunereaction in a host.

A T cell described herein can be, e.g., engineered such that it does notexpress a functional HLA on its surface. For example, a T cell describedherein, can be engineered such that cell surface expression HLA, e.g.,HLA class 1 and/or HLA class II, is downregulated. In some embodiments,downregulation of HLA may be accomplished by reducing or eliminatingexpression of beta-2 microglobulin (B2M).

In some embodiments, the T cell can lack a functional TCR and afunctional HLA, e.g., HLA class I and/or HLA class II.

Modified T cells that lack expression of a functional TCR and/or HLA canbe obtained by any suitable means, including a knock out or knock downof one or more subunit of TCR or HLA. For example, the T cell caninclude a knock down of TCR and/or HLA using siRNA, shRNA, clusteredregularly interspaced short palindromic repeats (CRISPR)transcription-activator like effector nuclease (TALEN), or zinc fingerendonuclease (ZFN).

In some embodiments, the allogeneic cell can be a cell which does notexpress or expresses at low levels an inhibitory molecule, e.g. by anymethod described herein. For example, the cell can be a cell that doesnot express or expresses at low levels an inhibitory molecule, e.g.,that can decrease the ability of a CAR-expressing cell to mount animmune effector response. Examples of inhibitory molecules include PD1,PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAGS, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GALS, adenosine, and TGFR (e.g., TGFRbeta).Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA,RNA or protein level, can optimize a CAR-expressing cell performance. Inembodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleicacid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularlyinterspaced short palindromic repeats (CRISPR), atranscription-activator like effector nuclease (TALEN), or a zinc fingerendonuclease (ZFN), e.g., as described herein, can be used.

siRNA and shRNA to Inhibit TCR or HLA

In some embodiments, TCR expression and/or HLA expression can beinhibited using siRNA or shRNA that targets a nucleic acid encoding aTCR and/or HLA, and/or an inhibitory molecule described herein (e.g.,PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86,B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHCclass I, MHC class II, GAL9, adenosine, and TGFR beta), in a cell, e.g.,T cell.

Expression systems for siRNA and shRNAs, and exemplary shRNAs, aredescribed, e.g., in paragraphs 649 and 650 of International ApplicationWO2015/142675, filed Mar. 13, 2015, which is incorporated by referencein its entirety.

CRISPR to Inhibit TCR or HLA

“CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/orHLA” as used herein refers to a set of clustered regularly interspacedshort palindromic repeats, or a system comprising such a set of repeats.“Cas”, as used herein, refers to a CRISPR-associated protein. A“CRISPR/Cas” system refers to a system derived from CRISPR and Cas whichcan be used to silence or mutate a TCR and/or HLA gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta), in a cell, e.g., T cell.

The CRISPR/Cas system, and uses thereof, are described, e.g., inparagraphs 651-658 of International Application WO2015/142675, filedMar. 13, 2015, which is incorporated by reference in its entirety.

TALEN to Inhibit TCR and/or HLA

“TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/orTCR” refers to a transcription activator-like effector nuclease, anartificial nuclease which can be used to edit the HLA and/or TCR gene,and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3,VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta), in a cell, e.g., T cell.

TALENs, and uses thereof, are described, e.g., in paragraphs 659-665 ofInternational Application WO2015/142675, filed Mar. 13, 2015, which isincorporated by reference in its entirety.

Zinc Finger Nuclease to Inhibit HLA and/or TCR

“ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN toinhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificialnuclease which can be used to edit the HLA and/or TCR gene, and/or aninhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4,TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GAL9, adenosine, and TGFR beta), in a cell, e.g., T cell.

ZFNs, and uses thereof, are described, e.g., in paragraphs 666-671 ofInternational Application WO2015/142675, filed Mar. 13, 2015, which isincorporated by reference in its entirety.

Telomerase Expression

Telomeres play a crucial role in somatic cell persistence, and theirlength is maintained by telomerase (TERT). Telomere length in CLL cellsmay be very short (Roth et al., “Significantly shorter telomeres inT-cells of patients with ZAP-70+/CD38 chronic lymphocytic leukaemia”British Journal of Haematology, 143, 383-386, Aug. 28, 2008), and may beeven shorter in manufactured CAR-expressing cells, e.g., CART19 cells,limiting their potential to expand after adoptive transfer to a patient.Telomerase expression can rescue CAR-expressing cells from replicativeexhaustion.

While not wishing to be bound by any particular theory, in someembodiments, a therapeutic T cell has short term persistence in apatient, due to shortened telomeres in the T cell; accordingly,transfection with a telomerase gene can lengthen the telomeres of the Tcell and improve persistence of the T cell in the patient. See CarlJune, “Adoptive T cell therapy for cancer in the clinic”, Journal ofClinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, animmune effector cell, e.g., a T cell, ectopically expresses a telomerasesubunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g.,hTERT. In some aspects, this disclosure provides a method of producing aCAR-expressing cell, comprising contacting a cell with a nucleic acidencoding a telomerase subunit, e.g., the catalytic subunit oftelomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with thenucleic acid before, simultaneous with, or after being contacted with aconstruct encoding a CAR.

Telomerase expression may be stable (e.g., the nucleic acid mayintegrate into the cell's genome) or transient (e.g., the nucleic aciddoes not integrate, and expression declines after a period of time,e.g., several days). Stable expression may be accomplished bytransfecting or transducing the cell with DNA encoding the telomerasesubunit and a selectable marker, and selecting for stable integrants.Alternatively or in combination, stable expression may be accomplishedby site-specific recombination, e.g., using the Cre/Lox or FLP/FRTsystem.

Transient expression may involve transfection or transduction with anucleic acid, e.g., DNA or RNA such as mRNA. In some embodiments,transient mRNA transfection avoids the genetic instability sometimesassociated with stable transfection with TERT. Transient expression ofexogenous telomerase activity is described, e.g., in InternationalApplication WO2014/130909, which is incorporated by reference herein inits entirety. In embodiments, mRNA-based transfection of a telomerasesubunit is performed according to the messenger RNA Therapeutics™platform commercialized by Moderna Therapeutics. For instance, themethod may be a method described in U.S. Pat. Nos. 8,710,200, 8,822,663,8,680,069, 8,754,062, 8,664,194, or 8680069.

In an embodiment, hTERT has the amino acid sequence of GenBank ProteinID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human TelomeraseCatalytic Subunit Gene, Is Up-Regulated in Tumor Cells and duringImmortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795):

(SEQ ID NO: 108) MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTERREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLEGVLRLKCHSLELDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPA LPSDFKTILD

In an embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 108. In anembodiment, the hTERT has a sequence of SEQ ID NO: 108. In anembodiment, the hTERT comprises a deletion (e.g., of no more than 5, 10,15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both.In an embodiment, the hTERT comprises a transgenic amino acid sequence(e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at theN-terminus, the C-terminus, or both.

In an embodiment, the hTERT is encoded by the nucleic acid sequence ofGenBank Accession No. AF018167 (Meyerson et al., “hEST2, the PutativeHuman Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cellsand during Immortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages785-795):

(SEQ ID NO: 23)    1caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc   61cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc  121tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg  181gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg  241cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg  301cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg  361cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct  421acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc  481gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg  541tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca  601ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg  661cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga  721ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg  781ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga  841cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag  901ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc  961agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc 1021ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 1081ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 1141agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 1201tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1261agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 1321cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg 1381acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt 1441acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 1501acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 1561agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1621ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 1681ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 1741atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga 1801gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt 1861cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc 1921gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1981ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt 2041tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2101gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc 2161cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc 2221aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 2281gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2341acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2401agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 2461gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg 2521gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 2581gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc 2641tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2701ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 2761agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga 2821tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 2881tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc 2941gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3001gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 3061acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 3121atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc 3181tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 3241ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3301tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc 3361agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3421cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 3481agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 3541ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 3601gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 3661tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 3721agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3781cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 3841caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt 3901gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg 3961ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa 4021aaaaaaa

In an embodiment, the hTERT is encoded by a nucleic acid having asequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 23. In an embodiment, the hTERT is encoded bya nucleic acid of SEQ ID NO: 23.

Chimeric Antigen Receptor (CAR)

The present invention provides immune effector cells (e.g., T cells, NKcells) that are engineered to contain one or more CARs that direct theimmune effector cells to cancer. This is achieved through an antigenbinding domain on the CAR that is specific for a cancer associatedantigen. There are two classes of cancer associated antigens (tumorantigens) that can be targeted by the CARs described herein: (1) cancerassociated antigens that are expressed on the surface of cancer cells;and (2) cancer associated antigens that itself is intracellar, however,a fragment of such antigen (peptide) is presented on the surface of thecancer cells by MHC (major histocompatibility complex).

Accordingly, an immune effector cell, e.g., obtained by a methoddescribed herein, can be engineered to contain a CAR that target one ofthe following cancer associated antigens (tumor antigens): CD19, CD123,CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag,PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT,IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta,PRSS21, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1,EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2,gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA,o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR,GPRC5D, CXORF61, CD97, CD179a, ALK, Plysialic acid, PLAC1, GloboH,NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1,NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, MAGE-A1, MAGE A1, ETV6-AML,sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints,ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor,Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK,AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,intestinal carboxyl esterase, and mut hsp70-2.

Bispecific CARs

In an embodiment a multispecific antibody molecule is a bispecificantibody molecule. A bispecific antibody has specificity for no morethan two antigens. A bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence which has bindingspecificity for a first epitope and a second immunoglobulin variabledomain sequence that has binding specificity for a second epitope. In anembodiment the first and second epitopes are on the same antigen, e.g.,the same protein (or subunit of a multimeric protein). In an embodimentthe first and second epitopes overlap. In an embodiment the first andsecond epitopes do not overlap. In an embodiment the first and secondepitopes are on different antigens, e.g., different proteins (ordifferent subunits of a multimeric protein). In an embodiment abispecific antibody molecule comprises a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a first epitope and a heavy chain variable domainsequence and a light chain variable domain sequence which have bindingspecificity for a second epitope. In an embodiment a bispecific antibodymolecule comprises a half antibody having binding specificity for afirst epitope and a half antibody having binding specificity for asecond epitope. In an embodiment a bispecific antibody moleculecomprises a half antibody, or fragment thereof, having bindingspecificity for a first epitope and a half antibody, or fragmentthereof, having binding specificity for a second epitope. In anembodiment a bispecific antibody molecule comprises a scFv, or fragmentthereof, have binding specificity for a first epitope and a scFv, orfragment thereof, have binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multi-specific (e.g.,a bispecific or a trispecific) antibody molecule. Protocols forgenerating bispecific or heterodimeric antibody molecules, and variousconfigurations for bispecific antibody molecules, are described in,e.g., paragraphs 455-458 of WO2015/142675, filed Mar. 13, 2015, which isincorporated by reference in its entirety.

In one aspect, the bispecific antibody molecule is characterized by afirst immunoglobulin variable domain sequence, e.g., a scFv, which hasbinding specificity for CD19, e.g., comprises a scFv as describedherein, or comprises the light chain CDRs and/or heavy chain CDRs from ascFv described herein, and a second immunoglobulin variable domainsequence that has binding specificity for a second epitope on adifferent antigen.

Chimeric TCR

In one aspect, the antibodies and antibody fragments of the presentinvention (e.g., CD19 antibodies and fragments) can be grafted to one ormore constant domain of a T cell receptor (“TCR”) chain, for example, aTCR alpha or TCR beta chain, to create a chimeric TCR. Without beingbound by theory, it is believed that chimeric TCRs will signal throughthe TCR complex upon antigen binding. For example, an scFv as disclosedherein, can be grafted to the constant domain, e.g., at least a portionof the extracellular constant domain, the transmembrane domain and thecytoplasmic domain, of a TCR chain, for example, the TCR alpha chainand/or the TCR beta chain. As another example, an antibody fragment, forexample a VL domain as described herein, can be grafted to the constantdomain of a TCR alpha chain, and an antibody fragment, for example a VHdomain as described herein, can be grafted to the constant domain of aTCR beta chain (or alternatively, a VL domain may be grafted to theconstant domain of the TCR beta chain and a VH domain may be grafted toa TCR alpha chain). As another example, the CDRs of an antibody orantibody fragment may be grafted into a TCR alpha and/or beta chain tocreate a chimeric TCR. For example, the LCDRs disclosed herein may begrafted into the variable domain of a TCR alpha chain and the HCDRsdisclosed herein may be grafted to the variable domain of a TCR betachain, or vice versa. Such chimeric TCRs may be produced, e.g., bymethods known in the art (For example, Willemsen R A et al, Gene Therapy2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496;Aggen et al, Gene Ther. 2012 April; 19(4):365-74).

Non-Antibody Scaffolds

In embodiments, the antigen binding domain comprises a non-antibodyscaffold, e.g., a fibronectin, ankyrin, domain antibody, lipocalin,small modular immuno-pharmaceutical, maxybody, Protein A, or affilin.The non-antibody scaffold has the ability to bind to target antigen on acell. In embodiments, the antigen binding domain is a polypeptide orfragment thereof of a naturally occurring protein expressed on a cell.In some embodiments, the antigen binding domain comprises a non-antibodyscaffold. A wide variety of non-antibody scaffolds can be employed solong as the resulting polypeptide includes at least one binding regionwhich specifically binds to the target antigen on a target cell.

Non-antibody scaffolds include: fibronectin (Novartis, Mass.), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

In an embodiment the antigen binding domain comprises the extracellulardomain, or a counter-ligand binding fragment thereof, of molecule thatbinds a counterligand on the surface of a target cell.

The immune effector cells can comprise a recombinant DNA constructcomprising sequences encoding a CAR, wherein the CAR comprises anantigen binding domain (e.g., antibody or antibody fragment, TCR or TCRfragment) that binds specifically to a tumor antigen, e.g., an tumorantigen described herein, and an intracellular signaling domain. Theintracellular signaling domain can comprise a costimulatory signalingdomain and/or a primary signaling domain, e.g., a zeta chain. Asdescribed elsewhere, the methods described herein can includetransducing a cell, e.g., from the population of T regulatory-depletedcells, with a nucleic acid encoding a CAR, e.g., a CAR described herein.

In specific aspects, a CAR comprises a scFv domain, wherein the scFv maybe preceded by an optional leader sequence such as provided in SEQ IDNO: 1, and followed by an optional hinge sequence such as provided inSEQ ID NO:2 or SEQ ID NO:36 or SEQ ID NO:38, a transmembrane region suchas provided in SEQ ID NO:6, an intracellular signalling domain thatincludes SEQ ID NO:7 or SEQ ID NO:16 and a CD3 zeta sequence thatincludes SEQ ID NO:9 or SEQ ID NO:10, e.g., wherein the domains arecontiguous with and in the same reading frame to form a single fusionprotein.

In one aspect, an exemplary CAR constructs comprise an optional leadersequence (e.g., a leader sequence described herein), an extracellularantigen binding domain (e.g., an antigen binding domain describedherein), a hinge (e.g., a hinge region described herein), atransmembrane domain (e.g., a transmembrane domain described herein),and an intracellular stimulatory domain (e.g., an intracellularstimulatory domain described herein). In one aspect, an exemplary CARconstruct comprises an optional leader sequence (e.g., a leader sequencedescribed herein), an extracellular antigen binding domain (e.g., anantigen binding domain described herein), a hinge (e.g., a hinge regiondescribed herein), a transmembrane domain (e.g., a transmembrane domaindescribed herein), an intracellular costimulatory signaling domain(e.g., a costimulatory signaling domain described herein) and/or anintracellular primary signaling domain (e.g., a primary signaling domaindescribed herein).

An exemplary leader sequence is provided as SEQ ID NO: 1. An exemplaryhinge/spacer sequence is provided as SEQ ID NO: 2 or SEQ ID NO:36 or SEQID NO:38. An exemplary transmembrane domain sequence is provided as SEQID NO:6. An exemplary sequence of the intracellular signaling domain ofthe 4-1BB protein is provided as SEQ ID NO: 7. An exemplary sequence ofthe intracellular signaling domain of CD27 is provided as SEQ ID NO:16.An exemplary CD3zeta domain sequence is provided as SEQ ID NO: 9 or SEQID NO:10.

In one aspect, the immune effector cell comprises a recombinant nucleicacid construct comprising a nucleic acid molecule encoding a CAR,wherein the nucleic acid molecule comprises a nucleic acid sequenceencoding an antigen binding domain, wherein the sequence is contiguouswith and in the same reading frame as the nucleic acid sequence encodingan intracellular signaling domain. An exemplary intracellular signalingdomain that can be used in the CAR includes, but is not limited to, oneor more intracellular signaling domains of, e.g., CD3-zeta, CD28, CD27,4-1BB, and the like. In some instances, the CAR can comprise anycombination of CD3-zeta, CD28, 4-1BB, and the like.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the nucleic acidmolecule, by deriving the nucleic acid molecule from a vector known toinclude the same, or by isolating directly from cells and tissuescontaining the same, using standard techniques. Alternatively, thenucleic acid of interest can be produced synthetically, rather thancloned.

Nucleic acids encoding a CAR can be introduced into the immune effectorcells using, e.g., a retroviral or lentiviral vector construct.

Nucleic acids encoding a CAR can also be introduced into the immuneeffector cell using, e.g., an RNA construct that can be directlytransfected into a cell. A method for generating mRNA for use intransfection involves in vitro transcription (IVT) of a template withspecially designed primers, followed by polyA addition, to produce aconstruct containing 3′ and 5′ untranslated sequence (“UTR”) (e.g., a 3′and/or 5′ UTR described herein), a 5′ cap (e.g., a 5′ cap describedherein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRESdescribed herein), the nucleic acid to be expressed, and a polyA tail,typically 50-2000 bases in length (e.g., described in the Examples,e.g., SEQ ID NO:35). RNA so produced can efficiently transfect differentkinds of cells. In one embodiment, the template includes sequences forthe CAR. In an embodiment, an RNA CAR vector is transduced into a cell,e.g., a T cell by electroporation.

Antigen Binding Domain

In one aspect, a plurality of the immune effector cells, e.g., thepopulation of T regulatory-depleted cells, include a nucleic acidencoding a CAR that comprises a target-specific binding elementotherwise referred to as an antigen binding domain. The choice ofbinding element depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state. Thus,examples of cell surface markers that may act as ligands for the antigenbinding domain in a CAR described herein include those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells.

In one aspect, the portion of the CAR comprising the antigen bindingdomain comprises an antigen binding domain that targets a tumor antigen,e.g., a tumor antigen described herein.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody, and a functional fragment thereof, including but not limitedto a single-domain antibody such as a heavy chain variable domain (VH),a light chain variable domain (VL) and a variable domain (VHH) ofcamelid derived nanobody, and to an alternative scaffold known in theart to function as antigen binding domain, such as a recombinantfibronectin domain, a T cell receptor (TCR), or a fragment there of,e.g., single chain TCR, and the like. In some instances, it isbeneficial for the antigen binding domain to be derived from the samespecies in which the CAR will ultimately be used in. For example, foruse in humans, it may be beneficial for the antigen binding domain ofthe CAR to comprise human or humanized residues for the antigen bindingdomain of an antibody or antibody fragment.

In an embodiment, the antigen binding domain comprises an anti-CD19antibody, or fragment thereof, e.g., an scFv. For example, the antigenbinding domain comprises a variable heavy chain and a variable lightchain listed in Table 1. The linker sequence joining the variable heavyand variable light chains can be, e.g., any of the linker sequencesdescribed herein, or alternatively, can be GSTSGSGKPGSGEGSTKG (SEQ IDNO:104).

TABLE 1 Anti-CD19 antibody binding domains CD19 huscFv1EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRL (SEQ IDHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKG NO: 39)GGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS CD19 huscFv2Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgip (SEQ IDarfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsg NO: 40)gggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgt lvtvss CD19huscFv3 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy(SEQ ID ssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvNO: 41) ssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqg tkleik CD19huscFv4 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy(SEQ ID qsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvNO: 42) ssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqg tkleik CD19huscFv5 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgip(SEQ ID arfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsgNO: 43) gggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtlvtvssCD19 huscFv6 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgip(SEQ ID arfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsgNO: 44) gggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtlvtvssCD19 huscFv7 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy(SEQ ID ssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvNO: 45) ssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikCD19 huscFv8 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy(SEQ ID qsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvNO: 46) ssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikCD19 huscFv9 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgip(SEQ ID arfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsgNO: 47) gggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdy wgqgtivtvssCD19 Hu QvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyscFv10 nsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtv(SEQ ID ssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqNO: 48) qkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik CD19 HuEivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgip scFv11arfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsg (SEQ IDgggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgse NO: 49)ttyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgt lvtvss CD19Hu Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyy scFv12nsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtv (SEQ IDssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgq NO: 50)aprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqg tkleik CD19muCTL019 Diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhsgvp(SEQ ID srfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsgNO: 51) gggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgt svtvss

TABLE 2 anti-CD19 antibody binding domains Antibody VH SequenceVL Sequence SSJ25-C1 QVQLLESGAELVRPGSSVKISCKASGYAFSSELVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVA YWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSG KFKGQATLTADKSSSTAYMQLSGLTSEDSAVTDFTLTITNVQSKDLADYFYFCQYNRYPYTSGGG YSCARKTISSVVDFYFDYWGQGTTVTTKLEIKRRS (SEQ ID NO: 4) (SEQ ID NO: 3)

Any CD19 CAR, e.g., the CD19 antigen binding domain of any known CD19CAR, can be used in accordance with the present disclosure. For example,LG-740; CD19 CAR described in the U.S. Pat. No. 8,399,645; U.S. Pat. No.7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260(2012); Cruz etal., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood,118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102(2010); Kochenderfer et al., Blood 122 (25):4129-39(2013); and 16th AnnuMeet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst10.

Exemplary target antigens that can be targeted using the CAR-expressingcells, include, but are not limited to, CD19, CD123, EGFRvIII,mesothelin, among others, as described in, for example, WO 2014/130635,WO 2014/130657, and WO 2015/090230, each of which is herein incorporatedby reference in its entirety.

In one embodiment, the CAR T cell that specifically binds to CD19 hasthe USAN designation TISAGENLECLEUCEL-T. CTL019 is made by a genemodification of T cells is mediated by stable insertion via transductionwith a self-inactivating, replication deficient Lentiviral (LV) vectorcontaining the CTL019 transgene under the control of the EF-1 alphapromoter. CTL019 can be a mixture of transgene positive and negative Tcells that are delivered to the subject on the basis of percenttransgene positive T cells.

In other embodiments, the CAR-expressing cells can specifically bind tohuman CD19, e.g., can include a CAR molecule, or an antigen bindingdomain (e.g., a humanized antigen binding domain) according to Table 3of WO2014/153270, incorporated herein by reference.

In other embodiments, the CAR-expressing cells can specifically bind toCD123, e.g., can include a CAR molecule (e.g., any of the CAR1-CARE), oran antigen binding domain according to Tables 1-2 of WO 2014/130635,incorporated herein by reference.

In other embodiments, the CAR-expressing cells can specifically bind toEGFRvIII, e.g., can include a CAR molecule, or an antigen binding domainaccording to Table 2 or SEQ ID NO:11 of WO 2014/130657, incorporatedherein by reference.

In other embodiments, the CAR-expressing cells can specifically bind tomesothelin, e.g., can include a CAR molecule, or an antigen bindingdomain according to Tables 2-3 of WO 2015/090230, incorporated herein byreference.

In one embodiment, the antigen binding domain comprises one, two three(e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, froman antibody listed above, and/or one, two, three (e.g., all three) lightchain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.In one embodiment, the antigen binding domain comprises a heavy chainvariable region and/or a variable light chain region of an antibodylisted or described above.

In some embodiments, the tumor antigen is a tumor antigen described inInternational Application WO2015/142675, filed Mar. 13, 2015, which isherein incorporated by reference in its entirety. In some embodiments,the tumor antigen is chosen from one or more of: CD19; CD123; CD22;CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7,CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1);CD33; epidermal growth factor receptor variant III (EGFRvIII);ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factorreceptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4);CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2(Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growthfactor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase;prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M);Ephrin B2; fibroblast activation protein alpha (FAP); insulin-likegrowth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);glycoprotein 100 (gp100); oncogene fusion protein consisting ofbreakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGicp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-1a);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLEC12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

In one embodiment, the antigen binding domain comprises one, two three(e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, froman antibody listed above, and/or one, two, three (e.g., all three) lightchain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above.In one embodiment, the antigen binding domain comprises a heavy chainvariable region and/or a variable light chain region of an antibodylisted or described above.

In one aspect, the anti-tumor antigen binding domain is a fragment,e.g., a single chain variable fragment (scFv). In one aspect, the anti-acancer associate antigen as described herein binding domain is a Fv, aFab, a (Fab′)2, or a bi-functional (e.g. bi-specific) hybrid antibody(e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In oneaspect, the antibodies and fragments thereof of the invention binds acancer associate antigen as described herein protein with wild-type orenhanced affinity.

In some instances, scFvs can be prepared according to a method known inthe art (see, for example, Bird et al., (1988) Science 242:423-426 andHuston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFvmolecules can be produced by linking VH and VL regions together usingflexible polypeptide linkers. The scFv molecules comprise a linker(e.g., a Ser-Gly linker) with an optimized length and/or amino acidcomposition. The linker length can greatly affect how the variableregions of a scFv fold and interact. In fact, if a short polypeptidelinker is employed (e.g., between 5-10 amino acids) intrachain foldingis prevented. Interchain folding is also required to bring the twovariable regions together to form a functional epitope binding site. Forexamples of linker orientation and size see, e.g., Hollinger et al. 1993Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent ApplicationPublication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCTpublication Nos. WO2006/020258 and WO2007/024715, which are incorporatedherein by reference.

An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or moreamino acid residues between its VL and VH regions. The linker sequencemay comprise any naturally occurring amino acid. In some embodiments,the linker sequence comprises amino acids glycine and serine. In anotherembodiment, the linker sequence comprises sets of glycine and serinerepeats such as (Gly₄Ser)n, where n is a positive integer equal to orgreater than 1 (SEQ ID NO:25). In one embodiment, the linker can be(Gly₄Ser)₄ (SEQ ID NO:27) or (Gly₄Ser)₃ (SEQ ID NO:28). Variation in thelinker length may retain or enhance activity, giving rise to superiorefficacy in activity studies.

In another aspect, the antigen binding domain is a T cell receptor(“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR).Methods to make such TCRs are known in the art. See, e.g., Willemsen R Aet al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012)(references are incorporated herein by its entirety). For example, scTCRcan be engineered that contains the Vα and Vβ genes from a T cell clonelinked by a linker (e.g., a flexible peptide). This approach is veryuseful to cancer associated target that itself is intracellar, however,a fragment of such antigen (peptide) is presented on the surface of thecancer cells by MHC.

Transmembrane Domain

With respect to the transmembrane domain, in various embodiments, a CARcan be designed to comprise a transmembrane domain that is attached tothe extracellular domain of the CAR. A transmembrane domain can includeone or more additional amino acids adjacent to the transmembrane region,e.g., one or more amino acid associated with the extracellular region ofthe protein from which the transmembrane was derived (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region)and/or one or more additional amino acids associated with theintracellular region of the protein from which the transmembrane proteinis derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids ofthe intracellular region). In one aspect, the transmembrane domain isone that is associated with one of the other domains of the CAR. In someinstances, the transmembrane domain can be selected or modified by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins, e.g., tominimize interactions with other members of the receptor complex. In oneaspect, the transmembrane domain is capable of homodimerization withanother CAR on the cell surface of a CAR-expressing cell. In a differentaspect, the amino acid sequence of the transmembrane domain may bemodified or substituted so as to minimize interactions with the bindingdomains of the native binding partner present in the same CART.

The transmembrane domain may be derived either from a natural or from arecombinant source. Where the source is natural, the domain may bederived from any membrane-bound or transmembrane protein. In one aspectthe transmembrane domain is capable of signalling to the intracellulardomain(s) whenever the CAR has bound to a target. A transmembrane domainof particular use in this invention may include at least thetransmembrane region(s) of e.g., the alpha, beta or zeta chain of theT-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In someembodiments, a transmembrane domain may include at least thetransmembrane region(s) of, e.g., KIR2DS2, OX40, CD2, CD27, LFA-1(CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rbeta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6,VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM,CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2,DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1,CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, PAG/Cbp, NKG2D, NKG2C, or CD19.

In some instances, the transmembrane domain can be attached to theextracellular region of the CAR, e.g., the antigen binding domain of theCAR, via a hinge, e.g., a hinge from a human protein. For example, inone embodiment, the hinge can be a human Ig (immunoglobulin) hinge,e.g., an IgG4 hinge, or a CD8a hinge. In one embodiment, the hinge orspacer comprises (e.g., consists of) the amino acid sequence of SEQ IDNO:2. In one aspect, the transmembrane domain comprises (e.g., consistsof) a transmembrane domain of SEQ ID NO: 6.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequenceESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:36).In some embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence ofGAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO:37).

In one aspect, the hinge or spacer comprises an IgD hinge. For example,in one embodiment, the hinge or spacer comprises a hinge of the aminoacid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO:38). Insome embodiments, the hinge or spacer comprises a hinge encoded by anucleotide sequence ofAGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACG TGACTGACCATT(SEQ ID NO:103).

In one aspect, the transmembrane domain may be recombinant, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. In one aspect a triplet of phenylalanine, tryptophan andvaline can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 10 aminoacids in length may form the linkage between the transmembrane domainand the cytoplasmic region of the CAR. A glycine-serine doublet providesa particularly suitable linker. For example, in one aspect, the linkercomprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 5). In someembodiments, the linker is encoded by a nucleotide sequence ofGGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 8).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Cytoplasmic Domain

The cytoplasmic domain or region of the CAR includes an intracellularsignaling domain. An intracellular signaling domain is generallyresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been introduced.

Examples of intracellular signaling domains for use in a CAR describedherein include the cytoplasmic sequences of the T cell receptor (TCR)and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any recombinant sequence that has thesame functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondaryand/or costimulatory signal is also required. Thus, T cell activationcan be said to be mediated by two distinct classes of cytoplasmicsignaling sequences: those that initiate antigen-dependent primaryactivation through the TCR (primary intracellular signaling domains) andthose that act in an antigen-independent manner to provide a secondaryor costimulatory signal (secondary cytoplasmic domain, e.g., acostimulatory domain).

A primary signaling domain regulates primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primaryintracellular signaling domains that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary intracellular signaling domains thatare of particular use in the invention include those of TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, CD278 (also known as “ICOS”), FcεRI, DAP10, DAP12, and CD66d. Inone embodiment, a CAR of the invention comprises an intracellularsignaling domain, e.g., a primary signaling domain of CD3-zeta, e.g., aCD3-zeta sequence described herein.

In one embodiment, a primary signaling domain comprises a modified ITAMdomain, e.g., a mutated ITAM domain which has altered (e.g., increasedor decreased) activity as compared to the native ITAM domain. In oneembodiment, a primary signaling domain comprises a modifiedITAM-containing primary intracellular signaling domain, e.g., anoptimized and/or truncated ITAM-containing primary intracellularsignaling domain. In an embodiment, a primary signaling domain comprisesone, two, three, four or more ITAM motifs.

Costimulatory Signaling Domain

The intracellular signalling domain of the CAR can comprise the CD3-zetasignaling domain by itself or it can be combined with any other desiredintracellular signaling domain(s) useful in the context of a CAR of theinvention. For example, the intracellular signaling domain of the CARcan comprise a CD3 zeta chain portion and a costimulatory signalingdomain. The costimulatory signaling domain refers to a portion of theCAR comprising the intracellular domain of a costimulatory molecule. Inone embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. In oneaspect, the intracellular domain is designed to comprise the signalingdomain of CD3-zeta and the signaling domain of ICOS.

A costimulatory molecule can be a cell surface molecule other than anantigen receptor or its ligands that is required for an efficientresponse of lymphocytes to an antigen. Examples of such moleculesinclude CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83, and thelike. For example, CD27 costimulation has been demonstrated to enhanceexpansion, effector function, and survival of human CART cells in vitroand augments human T cell persistence and antitumor activity in vivo(Song et al. Blood. 2012; 119(3):696-706). Further examples of suchcostimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR),SLAMF7, NKp80 (KLRF1), NKp30, NKp44, NKp46, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, NKG2D,NKG2C and PAG/Cbp.

The intracellular signaling sequences within the cytoplasmic portion ofthe CAR may be linked to each other in a random or specified order.Optionally, a short oligo- or polypeptide linker, for example, between 2and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) inlength may form the linkage between intracellular signaling sequences.In one embodiment, a glycine-serine doublet can be used as a suitablelinker. In one embodiment, a single amino acid, e.g., an alanine, aglycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed tocomprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signalingdomains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more,costimulatory signaling domains, are separated by a linker molecule,e.g., a linker molecule described herein. In one embodiment, theintracellular signaling domain comprises two costimulatory signalingdomains. In some embodiments, the linker molecule is a glycine residue.In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD28. In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain of4-1BB. In one aspect, the signaling domain of 4-1BB is a signalingdomain of SEQ ID NO: 7. In one aspect, the signaling domain of CD3-zetais a signaling domain of SEQ ID NO: 9.

In one aspect, the intracellular signaling domain is designed tocomprise the signaling domain of CD3-zeta and the signaling domain ofCD27. In one aspect, the signaling domain of CD27 comprises an aminoacid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQID NO:16). In one aspect, the signalling domain of CD27 is encoded by anucleic acid sequence ofAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCA GCCTATCGCTCC(SEQ ID NO:14).

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target or a different target(e.g., a target other than a cancer associated antigen described hereinor a different cancer associated antigen described herein, e.g., CD19,CD33, CLL-1, CD34, FLT3, or folate receptor beta). In one embodiment,the second CAR includes an antigen binding domain to a target expressedthe same cancer cell type as the cancer associated antigen. In oneembodiment, the CAR-expressing cell comprises a first CAR that targets afirst antigen and includes an intracellular signaling domain having acostimulatory signaling domain but not a primary signaling domain, and asecond CAR that targets a second, different, antigen and includes anintracellular signaling domain having a primary signaling domain but nota costimulatory signaling domain. While not wishing to be bound bytheory, placement of a costimulatory signaling domain, e.g., 4-1BB,CD28, ICOS, CD27 or OX-40, onto the first CAR, and the primary signalingdomain, e.g., CD3 zeta, on the second CAR can limit the CAR activity tocells where both targets are expressed. In one embodiment, the CARexpressing cell comprises a first cancer associated antigen CAR thatincludes an antigen binding domain that binds a target antigen describedherein, a transmembrane domain and a costimulatory domain and a secondCAR that targets a different target antigen (e.g., an antigen expressedon that same cancer cell type as the first target antigen) and includesan antigen binding domain, a transmembrane domain and a primarysignaling domain. In another embodiment, the CAR expressing cellcomprises a first CAR that includes an antigen binding domain that bindsa target antigen described herein, a transmembrane domain and a primarysignaling domain and a second CAR that targets an antigen other than thefirst target antigen (e.g., an antigen expressed on the same cancer celltype as the first target antigen) and includes an antigen binding domainto the antigen, a transmembrane domain and a costimulatory signalingdomain.

In another aspect, the disclosure features a population ofCAR-expressing cells, e.g., CART cells. In some embodiments, thepopulation of CAR-expressing cells comprises a mixture of cellsexpressing different CARs.

For example, in one embodiment, the population of CART cells can includea first cell expressing a CAR having an antigen binding domain to acancer associated antigen described herein, and a second cell expressinga CAR having a different antigen binding domain, e.g., an antigenbinding domain to a different a cancer associated antigen describedherein, e.g., an antigen binding domain to a cancer associated antigendescribed herein that differs from the cancer associate antigen bound bythe antigen binding domain of the CAR expressed by the first cell.

As another example, the population of CAR-expressing cells can include afirst cell expressing a CAR that includes an antigen binding domain to acancer associated antigen described herein, and a second cell expressinga CAR that includes an antigen binding domain to a target other than acancer associate antigen as described herein. In one embodiment, thepopulation of CAR-expressing cells includes, e.g., a first cellexpressing a CAR that includes a primary intracellular signaling domain,and a second cell expressing a CAR that includes a secondary signalingdomain.

In another aspect, the disclosure features a population of cells whereinat least one cell in the population expresses a CAR having an antigenbinding domain to a cancer associated antigen described herein, and asecond cell expressing another agent, e.g., an agent which enhances theactivity of a CAR-expressing cell. For example, in one embodiment, theagent can be an agent which inhibits an inhibitory molecule. Inhibitorymolecules, e.g., PD-1, can, in some embodiments, decrease the ability ofa CAR-expressing cell to mount an immune effector response. Examples ofinhibitory molecules include PD-1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 orCD270), KIR, A2aR, MHC class I, MHC class II, GALS, adenosine, and TGFR(e.g., TGFRbeta). In one embodiment, the agent which inhibits aninhibitory molecule comprises a first polypeptide, e.g., an inhibitorymolecule, associated with a second polypeptide that provides a positivesignal to the cell, e.g., an intracellular signaling domain describedherein. In one embodiment, the agent comprises a first polypeptide,e.g., of an inhibitory molecule such as PD-1, PD-L1, CTLA4, TIM3, CEACAM(CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1,CD160, 2B4 and TGFR beta, or a fragment of any of these, and a secondpolypeptide which is an intracellular signaling domain described herein(e.g., comprising a costimulatory domain (e.g., 41BB, CD27, OX40 orCD28, e.g., as described herein) and/or a primary signaling domain(e.g., a CD3 zeta signaling domain described herein). In one embodiment,the agent comprises a first polypeptide of PD-1 or a fragment thereof,and a second polypeptide of an intracellular signaling domain describedherein (e.g., a CD28 signaling domain described herein and/or a CD3 zetasignaling domain described herein).

The sequences of anti-CD19 binding domains are provided herein inTable 1. Full CAR constructs can be generated using any of the antigenbinding domains described in Table 1 with one or more additional CARcomponent provided below.

leader (amino acid sequence)  (SEQ ID NO: 1) MALPVTALLLPLALLLHAARPleader (nucleic acid sequence)  (SEQ ID NO: 12)ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGCA TGCCGCTAGACCCCD8 hinge (amino acid sequence)  (SEQ ID NO: 2)QPLSLRPEACRPAAGGAVHTRGLDFACD CD8 hinge (nucleic acid sequence) (SEQ ID NO: 13) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATCD8 transmembrane (amino acid sequence)  (SEQ ID NO: 6)IYIWAPLAGTCGVLLLSLVITLYC transmembrane (nucleic acid sequence) (SEQ ID NO: 17) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC 4-1BB Intracellular domain (amino acid sequence) (SEQ ID NO: 7) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL4-1BB Intracellular domain (nucleic acid sequence)  (SEQ ID NO: 18)AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD3 zeta domain (amino acid sequence) (SEQ ID NO: 9) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRCD3 zeta (nucleic acid sequence)  (SEQ ID NO: 20)AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCD3 zeta domain (amino acid sequence; NCBI Reference Sequence NM_000734.3)  (SEQ ID NO: 10)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPRCD3 zeta (nucleic acid sequence; NCBI Reference  Sequence NM_000734.3); (SEQ ID NO: 21) agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc IgG4 Hinge (amino acid sequence) (SEQ ID NO: 36) ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM IgG4 Hinge (nucleotide sequence) (SEQ ID NO: 37) GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG EF1 alpha promoter(SEQ ID NO: 11) CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA. Gly/Ser  (SEQ ID NO: 25) GGGGSGly/Ser (SEQ ID NO: 26): This sequence may encompass 1-6 ″Gly Gly Gly Gly Ser″ repeating unitsGGGGSGGGGS GGGGSGGGGS GGGGSGGGGS Gly/Ser  (SEQ ID NO: 27)GGGGSGGGGS GGGGSGGGGS Gly/Ser  (SEQ ID NO: 28) GGGGSGGGGS GGGGS Gly/Ser (SEQ ID NO: 29) GGGS PolyA (SEQ ID NO: 30):  A5000PolyA (SEQ ID NO: 31):  A100 PolyT (SEQ ID NO: 32):  T5000PolyA (SEQ ID NO: 33):  A5000 PolyA (SEQ ID NO: 34):  A400PolyA (SEQ ID NO: 35):  A2000Gly/Ser (SEQ ID NO: 15): This sequence may encompass 1-10 ″Gly Gly Gly Ser″ repeating unitsGGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS

Exemplary CD19 CAR constructs that can be used in the methods describedherein are shown in Table 3:

TABLE 3 CD19 CAR Constructs Name SEQ ID Sequence CAR1 CAR1 scFv  39EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHT domainSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS 103101  52atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR1tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Solubleagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg scFv-nttatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagccaccaccatcatcaccatcaccat 103101  64 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR1yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Solublentlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs scFv-aagvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 104875  90atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 1-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104875  77MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasqdiskyln w CAR 1-yqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqg Full-aantlpyt fgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs gvslp dygvswirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslk lssvtaadtavyycakhyyyggsyamdy wgqgtivtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR2 CAR2 scFv  40eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs domaingiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 103102  53atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR2-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Solubleagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg scFv-nttatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagccaccaccatcatcaccatcaccat 103102  65 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR2-yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Solublentlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs scFv-aagvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvss hhhhhhhh 104876  91atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 2-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104876  78MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasgdiskyln w CAR 2-yqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqg Full-aantlpyt fgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs gyslp dyg v swirqppgkglewig viwgsettyyqsslks rvtiskdnsknqvslk lssvtaadtavyycakhyyyggsyamdy wgqgtivtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 3 CAR3 scFv  41qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domaintyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 103104  54atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 3-tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Solublectctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc scFv-nttggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaacatcaccaccatcatcaccatcac 103104  66 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygys CAR 3-wirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadta Solublevyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls scFv-aalspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104877  92atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 3-tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-ntctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104877  79MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpd ygvs CAR 3-wirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadta Full-aavyycak hyyyggsyamdy wgqgtivtvssggggsggggsggggseivmtqspatls lspgeratlscrasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsg tdytltisslqpedfavyfcqqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgylllslvitlyckrgrkkllyifkufmrpvqttcleedgcscrfpeeeeggcelrykfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglycclstatkdtydalhmcialppr CAR 4 CAR4 scFv  42qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domaintyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 103106  55atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR4-tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Solublectctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc scFv-nttggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaacatcaccaccatcatcaccatcac 103106  67 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR4-wirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadta Solublevyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls scFv-aalspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104878  93atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 4-tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full ntctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 104878  80MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs CAR 4-wirqppgkglewig viwgsettyygsslks rvtiskdnsknqvslklssvtaadta Full-aavyycak hyyyggsyamdy wgqgtlvtvssggggsggggsggggseivmtqspatls lspgeratlscrasgdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsg tdytltisslqpedfavyfcqqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 5 CAR5 scFv  43eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs domaingiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 99789  56atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgc CAR5-tcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcg Solubleagagggcaaccctttcatgcagggccagccaggacatttctaagtacctcaactgg scFv-nttatcagcagaagccagggcaggctcctcgcctgctgatctaccacaccagccgcctccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccctcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcaggggaatactctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcggaggatcaggcggtggcggaagcggaggaggtggctccggaggaggaggttcccaagtgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctgacttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgccagcctccggggaagggtcttgaatggattggggtgatttggggatcagagactacttactactcttcatcacttaagtcacgggtcaccatcagcaaagataatagcaagaaccaagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattgtgccaaacattactattacggagggtcttatgctatggactactggggacaggggaccctggtgactgtctctagccatcaccatcaccaccatcatcac 99789  68MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnw CAR5-yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Solublentlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl scFv-aatctvsgvslpdygvswirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 104879  94atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 5-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggcggaggcgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactactcttcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104879  81MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasgdiskyln w CAR 5-yqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqg Full-aantlpyt fgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl tctvsgvslpdygvs wirqppgkglewig viwgsettyyssslks rvtiskdnskn qvslklssvtaadtavyycakhyyyggsyamdy wgqgtivtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR6 CAR6  44eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs scFvgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs domainggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 99790  57atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgc CAR6-tcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcg Solubleagagggcaaccctttcatgcagggccagccaggacatttctaagtacctcaactgg scFv-nttatcagcagaagccagggcaggctcctcgcctgctgatctaccacaccagccgcctccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccctcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcaggggaatactctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcggaggatcaggcggtggcggaagcggaggaggtggctccggaggaggaggttcccaagtgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctgacttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgccagcctccggggaagggtcttgaatggattggggtgatttggggatcagagactacttactaccagtcatcacttaagtcacgggtcaccatcagcaaagataatagcaagaaccaagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattgtgccaaacattactattacggagggtcttatgctatggactactggggacaggggaccctggtgactgtctctagccatcaccatcaccaccatcatcac 99790  69MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnw CAR6-yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Solublentlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl scFv-aatctvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtilvtvss hhhhhhhh 104880  95atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR6-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggaggcggagggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104880  82MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasgdiskyln w CARE6-yqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqg Full-aantlpyt fgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl tctvsgvslpdygvs wirqppgkglewig viwgsettyygsslks rvtiskdnskn qvslklssvtaadtavyycakhyyyggsyamdy wgqgtivtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR7 CAR7 scFv  45qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domaintyyssslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 100796  58atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgc CAR7-caggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgaga Solublectctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtca scFv-nttggatccgccaacctcccgggaaagggcttgaatggattggtgtcatctggggttctgaaaccacctactactcatcttccctgaagtccagggtgaccatcagcaaggataattccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgccgtgtattactgcgccaagcactactattacggaggaagctacgctatggactattggggacagggcactctcgtgactgtgagcagcggcggtggagggtctggaggtggaggatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtcaccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccagccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctcgcctcctgatctaccatacctcacgccttcactctggtatccccgctcggtttagcggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaagatttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagggaaccaagctcgaaatcaagcaccatcaccatcatcaccaccat 100796  70MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR7-wirqppgkglewigviwgsettyyssslksrvtiskdnsknqvslklssvtaadta Solublevyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqs scFv-aapatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104881  96atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR7tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-ntctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactattcatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104881  83MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs CAR 7wirqppgkglewig viwgsettyyssslks rvtiskdnsknqvslklssvtaadta Full-aavyycak hyyyggsyamdy wgqgtlvtvssggggsggggsggggsggggseivmtqspatlslspgeratlsc rasgdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 8 CAR8 scFv  46qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset domaintyyqsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 100798  59atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgc CARS8-caggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgaga Solublectctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtca scFv-nttggatccgccaacctcccgggaaagggcttgaatggattggtgtcatctggggttctgaaaccacctactaccagtcttccctgaagtccagggtgaccatcagcaaggataattccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgccgtgtattactgcgccaagcactactattacggaggaagctacgctatggactattggggacagggcactctcgtgactgtgagcagcggcggtggagggtctggaggtggaggatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtcaccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccagccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctcgcctcctgatctaccatacctcacgccttcactctggtatccccgctcggtttagcggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaagatttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagggaaccaagctcgaaatcaagcaccatcaccatcatcatcaccac 100798  71MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvs CAR8-wirqppgkglewigviwgsettyyqsslksrvtiskdnsknqvslklssvtaadta Solublevyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivmtqs scFv-aapatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 104882  97atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 8-tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-ntctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactatcaatcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccggaggcggtgggtcagaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 104882  84MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslp dygvs CAR 8-wirqppgkglewig viwgsettyygsslks rvtiskdnsknqvslklssvtaadta Full-aavyycak hyyyggsyamdy wgqgtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlsc rasqdiskyln wyqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqgntlpyt fgqgtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR 9 CAR9 scFv  47eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs domaingiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 99789  60atggccctcccagtgaccgctctgctgctgcctctcgcacttcttctccatgccgc CAR9-tcggcctgagatcgtcatgacccaaagccccgctaccctgtccctgtcacccggcg Solubleagagggcaaccctttcatgcagggccagccaggacatttctaagtacctcaactgg scFv-nttatcagcagaagccagggcaggctcctcgcctgctgatctaccacaccagccgcctccacagcggtatccccgccagattttccgggagcgggtctggaaccgactacaccctcaccatctcttctctgcagcccgaggatttcgccgtctatttctgccagcaggggaatactctgccgtacaccttcggtcaaggtaccaagctggaaatcaagggaggcggaggatcaggcggtggcggaagcggaggaggtggctccggaggaggaggttcccaagtgcagcttcaagaatcaggacccggacttgtgaagccatcagaaaccctctccctgacttgtaccgtgtccggtgtgagcctccccgactacggagtctcttggattcgccagcctccggggaagggtcttgaatggattggggtgatttggggatcagagactacttactacaattcatcacttaagtcacgggtcaccatcagcaaagataatagcaagaaccaagtgtcacttaagctgtcatctgtgaccgccgctgacaccgccgtgtactattgtgccaaacattactattacggagggtcttatgctatggactactggggacaggggaccctggtgactgtctctagccatcaccatcaccaccatcatcac 99789  72MALPVTALLLPLALLLHAARP eivmtqspatlslspgeratlscrasqdiskylnw CAR9-yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Solublentlpytfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl scFv-aatctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 105974  98atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 9-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggaggcggtgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105974  85MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlsc rasgdiskyln w CAR 9-yqqkpgqaprlliy htsrlhs giparfsgsgsgtdytltisslqpedfavyfc qqg Full-aantlpyt fgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpsetlsl tctvsgvslpdygvs wirqppgkglewig viwgsettyynsslks rvtiskdnskn qvslklssvtaadtavyycakhyyyggsyamdy wgqgtivtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CAR10 CAR10  48qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset scFvtyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq domaingtivtvssggggsggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 100796  61atggcactgcctgtcactgccctcctgctgcctctggccctccttctgcatgccgc CAR10-caggccccaagtccagctgcaagagtcaggacccggactggtgaagccgtctgaga Solublectctctcactgacttgtaccgtcagcggcgtgtccctccccgactacggagtgtca scFv-nttggatccgccaacctcccgggaaagggcttgaatggattggtgtcatctggggttctgaaaccacctactacaactcttccctgaagtccagggtgaccatcagcaaggataattccaagaaccaggtcagccttaagctgtcatctgtgaccgctgctgacaccgccgtgtattactgcgccaagcactactattacggaggaagctacgctatggactattggggacagggcactctcgtgactgtgagcagcggcggtggagggtctggaggtggaggatccggtggtggtgggtcaggcggaggagggagcgagattgtgatgactcagtcaccagccaccctttctctttcacccggcgagagagcaaccctgagctgtagagccagccaggacatttctaagtacctcaactggtatcagcaaaaaccggggcaggcccctcgcctcctgatctaccatacctcacgccttcactctggtatccccgctcggtttagcggatcaggatctggtaccgactacactctgaccatttccagcctgcagccagaagatttcgcagtgtatttctgccagcagggcaatacccttccttacaccttcggtcagggaaccaagctcgaaatcaagcaccatcaccatcatcaccaccat 100796  73MALPVTALLLPLALLLHAARP qvqlqesgpglvkpsetlsltctvsgvslpdygvs CVOR10 -wirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadta Solublevyycakhyyyggsyamdywgqgtivtvssggggsggggsggggsggggseivmtqs scFv-aapatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 105975  99atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 10tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagcggaggcggtgggagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105975  86MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSC RASQDISKYLN W CAR 10YQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQG Full-aaNTLPYT FGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSL TCTVSGVSLPDYGVS WIRQPPGKGLEWIG VIWGSETTYYNSSLKS RVTISKDNSKN QVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR11 CAR11  49eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhs scFvgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleikggggs domainggggsggggsqvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss 103101  62Atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR11-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Solubleagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattgg scFv-nttatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaattcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagccaccaccatcatcaccatcaccat 103101  74 MALPVTALLLPLALLLHAARPeivmtqspatlslspgeratlscrasqdiskylnw CAR11-yqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqg Solublentlpytfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltctvs scFv-aagvslpdygvswirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtvss hhhhhhhh 105976 100atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR 11tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Full-ntctctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagctggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccggaggtggcggaagcgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaaaccactactcccgctccaaggccacccacccctgccccgaccatcgcctctcagccgctttccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg 105976  87MALPVTALLLPLALLLRAARPQVQLQESGPGLVKPSETLSLTCTVSGVSLP DYGVS CAR 11WIRQPPGKGLEWIG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLKLSSVTAADTA Full-aaVYYCAK HYYYGGSYAMDY WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCR ASQDISKYLN WYQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQGNTLPYT FGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CAR12 CAR12  50qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewigviwgset scFvtyynsslksrvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgq domaingtivtvssggggsggggsggggseivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik 103104  63atggctctgcccgtgaccgcactcctcctgccactggctctgctgcttcacgccgc CAR12 -tcgcccacaagtccagcttcaagaatcagggcctggtctggtgaagccatctgaga Solublectctgtccctcacttgcaccgtgagcggagtgtccctcccagactacggagtgagc scFv-nttggattagacagcctcccggaaagggactggagtggatcggagtgatttggggtagcgaaaccacttactataactcttccctgaagtcacgggtcaccatttcaaaggataactcaaagaatcaagtgagcctcaagctctcatcagtcaccgccgctgacaccgccgtgtattactgtgccaagcattactactatggagggtcctacgccatggactactggggccagggaactctggtcactgtgtcatctggtggaggaggtagcggaggaggcgggagcggtggaggtggctccgaaatcgtgatgacccagagccctgcaaccctgtccctttctcccggggaacgggctaccctttcttgtcgggcatcacaagatatctcaaaatacctcaattggtatcaacagaagccgggacaggcccctaggcttcttatctaccacacctctcgcctgcatagcgggattcccgcacgctttagcgggtctggaagcgggaccgactacactctgaccatctcatctctccagcccgaggacttcgccgtctacttctgccagcagggtaacaccctgccgtacaccttcggccagggcaccaagcttgagatcaaacatcaccaccatcatcaccatcac 103104  75 MALPVTALLLPLALLLHAARPqvqlqesgpglvkpsetlsltctvsgvslpdygys CAR12 -wirqppgkglewigviwgsettyynsslksrvtiskdnsknqvslklssvtaadta Solublevyycakhyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspatls scFv -aalspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkleik hhhhhhhh 105977 101atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgc CAR 12-tcggcccgaaattgtgatgacccagtcacccgccactcttagcctttcacccggtg Full-ntagcgcgcaaccctgtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccggacaggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggttcagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagaggacttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcaccaagctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagccaggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactgacttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccggggaagggtctggaatggattggagtgatttggggctctgagactacttactacaactcatccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaactgtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcgggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactaccccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtccggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctgcgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatcactctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgaggcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggaggaaggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagcaggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgctggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatccccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgagattggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactcagcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctc gg 105977  88MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSC RASQDISKYLN W CAR 12-YQQKPGQAPRLLIY HTSRLHS GIPARFSGSGSGTDYTLTISSLQPEDFAVYFC QQG Full-aaNTLPYT FGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVS GVSLP DYGVSWIRQPPGKGLEWIG VIWGSETTYYNSSLKS RVTISKDNSKNQVSLK LSSVTAADTAVYYCAKHYYYGGSYAMDY WGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CTL019 CTL019-  22atggccctgcccgtcaccgctctgctgctgccccttgctctgcttcttcatgcagc Solubleaaggccggacatccagatgacccaaaccacctcatccctctctgcctctcttggag scFv-acagggtgaccatttcttgtcgcgccagccaggacatcagcaagtatctgaactgg Histag-nttatcagcagaagccggacggaaccgtgaagctcctgatctaccatacctctcgcctgcatagcggcgtgccctcacgcttctctggaagcggatcaggaaccgattattctctcactatttcaaatcttgagcaggaagatattgccacctatttctgccagcagggtaataccctgccctacaccttcggaggagggaccaagctcgaaatcaccggtggaggaggcagcggcggtggagggtctggtggaggtggttctgaggtgaagctgcaagaatcaggccctggacttgtggccccttcacagtccctgagcgtgacttgcaccgtgtccggagtctccctgcccgactacggagtgtcatggatcagacaacctccacggaaaggactggaatggctcggtgtcatctggggtagcgaaactacttactacaattcagccctcaaaagcaggctgactattatcaaggacaacagcaagtcccaagtctttcttaagatgaactcactccagactgacgacaccgcaatctactattgtgctaagcactactactacggaggatcctacgctatggattactggggacaaggtacttccgtcactgtctcttcacaccatcatcaccatcaccatcac CTL019-  76 MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnw Solubleyqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqg scFv-ntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvs Histag- gvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvss hhhhhhhh CTL019 102atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgc Full-ntcaggccggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctc gc CTL019  89MALPVTALLLPLALLLHAARPdiqmtqttsslsaslgdrvtiscrasqdiskylnw Full-aayqqkpdgtvklliyhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvsstttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttgeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr CTL019  51diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklliyhtsrlhs scFvgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpytfgggtkleitggggs domainggggsggggsevklqesgpglvapsqslsvtctvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltiikdnsksqvflkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtvssCo-Expression of CAR with Other Molecules or Agents

Co-Expression of a Second CAR

In one aspect, the CAR-expressing cell described herein can furthercomprise a second CAR, e.g., a second CAR that includes a differentantigen binding domain, e.g., to the same target (e.g., CD19) or adifferent target (e.g., a target other than CD19, e.g., a targetdescribed herein). In one embodiment, the CAR-expressing cell comprisesa first CAR that targets a first antigen and includes an intracellularsignaling domain having a costimulatory signaling domain but not aprimary signaling domain, and a second CAR that targets a second,different, antigen and includes an intracellular signaling domain havinga primary signaling domain but not a costimulatory signaling domain.Placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27,OX-40 or ICOS, onto the first CAR, and the primary signaling domain,e.g., CD3 zeta, on the second CAR can limit the CAR activity to cellswhere both targets are expressed. In one embodiment, the CAR expressingcell comprises a first CAR that includes an antigen binding domain, atransmembrane domain and a costimulatory domain and a second CAR thattargets another antigen and includes an antigen binding domain, atransmembrane domain and a primary signaling domain. In anotherembodiment, the CAR expressing cell comprises a first CAR that includesan antigen binding domain, a transmembrane domain and a primarysignaling domain and a second CAR that targets another antigen andincludes an antigen binding domain to the antigen, a transmembranedomain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing cell comprises an XCAR describedherein and an inhibitory CAR. In one embodiment, the inhibitory CARcomprises an antigen binding domain that binds an antigen found onnormal cells but not cancer cells, e.g., normal cells that also expressX. In one embodiment, the inhibitory CAR comprises the antigen bindingdomain, a transmembrane domain and an intracellular domain of aninhibitory molecule. For example, the intracellular domain of theinhibitory CAR can be an intracellular domain of PD1, PD-L1, PD-L2,CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5), LAGS, VISTA,BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4(VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II,GALS, adenosine, and TGFR (e.g., TGFRbeta).

In one embodiment, when the CAR-expressing cell comprises two or moredifferent CARs, the antigen binding domains of the different CARs can besuch that the antigen binding domains do not interact with one another.For example, a cell expressing a first and second CAR can have anantigen binding domain of the first CAR, e.g., as a fragment, e.g., anscFv, that does not form an association with the antigen binding domainof the second CAR, e.g., the antigen binding domain of the second CAR isa VHH.

In some embodiments, the antigen binding domain comprises a singledomain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, lamprey, fish,shark, goat, rabbit, and bovine. This term also includes naturallyoccurring single domain antibody molecules from species other thanCamelidae and sharks.

In one aspect, an SDAB molecule can be derived from a variable region ofthe immunoglobulin found in fish, such as, for example, that which isderived from the immunoglobulin isotype known as Novel Antigen Receptor(NAR) found in the serum of shark. Methods of producing single domainmolecules derived from a variable region of NAR (“IgNARs”) are describedin WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.

According to another aspect, an SDAB molecule is a naturally occurringsingle domain antigen binding molecule known as heavy chain devoid oflight chains. Such single domain molecules are disclosed in WO 9404678and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.For clarity reasons, this variable domain derived from a heavy chainmolecule naturally devoid of light chain is known herein as a VHH ornanobody to distinguish it from the conventional VH of four chainimmunoglobulins. Such a VHH molecule can be derived from Camelidaespecies, for example in camel, llama, dromedary, alpaca and guanaco.Other species besides Camelidae may produce heavy chain moleculesnaturally devoid of light chain; such VHHs are within the scope of theinvention.

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display).

It has also been discovered, that cells having a plurality of chimericmembrane embedded receptors comprising an antigen binding domain thatinteractions between the antigen binding domain of the receptors can beundesirable, e.g., because it inhibits the ability of one or more of theantigen binding domains to bind its cognate antigen. Accordingly,disclosed herein are cells having a first and a second non-naturallyoccurring chimeric membrane embedded receptor comprising antigen bindingdomains that minimize such interactions. Also disclosed herein arenucleic acids encoding a first and a second non-naturally occurringchimeric membrane embedded receptor comprising an antigen bindingdomains that minimize such interactions, as well as methods of makingand using such cells and nucleic acids. In an embodiment the antigenbinding domain of one of the first and the second non-naturallyoccurring chimeric membrane embedded receptor, comprises an scFv, andthe other comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence.

In some embodiments, a composition herein comprises a first and secondCAR, wherein the antigen binding domain of one of the first and thesecond CAR does not comprise a variable light domain and a variableheavy domain. In some embodiments, the antigen binding domain of one ofthe first and the second CAR is an scFv, and the other is not an scFv.In some embodiments, the antigen binding domain of one of the first andthe second CAR comprises a single VH domain, e.g., a camelid, shark, orlamprey single VH domain, or a single VH domain derived from a human ormouse sequence. In some embodiments, the antigen binding domain of oneof the first and the second CAR comprises a nanobody. In someembodiments, the antigen binding domain of one of the first and thesecond CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of the first andthe second CAR comprises an scFv, and the other comprises a single VHdomain, e.g., a camelid, shark, or lamprey single VH domain, or a singleVH domain derived from a human or mouse sequence. In some embodiments,the antigen binding domain of one of the first and the second CARcomprises an scFv, and the other comprises a nanobody. In someembodiments, the antigen binding domain of one of the first and thesecond CAR comprises an scFv, and the other comprises a camelid VHHdomain.

In some embodiments, when present on the surface of a cell, binding ofthe antigen binding domain of the first CAR to its cognate antigen isnot substantially reduced by the presence of the second CAR. In someembodiments, binding of the antigen binding domain of the first CAR toits cognate antigen in the presence of the second CAR is at least 85%,90%, 95%, 96%, 97%, 98% or 99%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or99% of binding of the antigen binding domain of the first CAR to itscognate antigen in the absence of the second CAR.

In some embodiments, when present on the surface of a cell, the antigenbinding domains of the first and the second CAR, associate with oneanother less than if both were scFv antigen binding domains. In someembodiments, the antigen binding domains of the first and the secondCAR, associate with one another at least 85%, 90%, 95%, 96%, 97%, 98% or99% less than, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% less than ifboth were scFv antigen binding domains.

Co-Expression of an Agent that Enhances CAR Activity

In another aspect, the CAR-expressing cell described herein can furtherexpress another agent, e.g., an agent that enhances the activity orfitness of a CAR-expressing cell.

For example, in one embodiment, the agent can be an agent which inhibitsa molecule that modulates or regulates, e.g., inhibits, T cell function.In some embodiments, the molecule that modulates or regulates T cellfunction is an inhibitory molecule. Inhibitory molecules, e.g., PD1,can, in some embodiments, decrease the ability of a CAR-expressing cellto mount an immune effector response. Examples of inhibitory moleculesinclude PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270),KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, or TGFR beta.

In embodiments, an agent, e.g., an inhibitory nucleic acid, e.g., adsRNA, e.g., an siRNA or shRNA; or e.g., an inhibitory protein orsystem, e.g., a clustered regularly interspaced short palindromicrepeats (CRISPR), a transcription-activator like effector nuclease(TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein,can be used to inhibit expression of a molecule that modulates orregulates, e.g., inhibits, T-cell function in the CAR-expressing cell.In an embodiment the agent is an shRNA, e.g., an shRNA described herein.In an embodiment, the agent that modulates or regulates, e.g., inhibits,T-cell function is inhibited within a CAR-expressing cell. For example,a dsRNA molecule that inhibits expression of a molecule that modulatesor regulates, e.g., inhibits, T-cell function is linked to the nucleicacid that encodes a component, e.g., all of the components, of the CAR.

In one embodiment, the agent which inhibits an inhibitory moleculecomprises a first polypeptide, e.g., an inhibitory molecule, associatedwith a second polypeptide that provides a positive signal to the cell,e.g., an intracellular signaling domain described herein. In oneembodiment, the agent comprises a first polypeptide, e.g., of aninhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA,TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM(TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9,adenosine, or TGFR beta, or a fragment of any of these (e.g., at least aportion of an extracellular domain of any of these), and a secondpolypeptide which is an intracellular signaling domain described herein(e.g., comprising a costimulatory domain (e.g., 41BB, CD27 or CD28,e.g., as described herein) and/or a primary signaling domain (e.g., aCD3 zeta signaling domain described herein). In one embodiment, theagent comprises a first polypeptide of PD1 or a fragment thereof (e.g.,at least a portion of an extracellular domain of PD1), and a secondpolypeptide of an intracellular signaling domain described herein (e.g.,a CD28 signaling domain described herein and/or a CD3 zeta signalingdomain described herein). PD1 is an inhibitory member of the CD28 familyof receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 isexpressed on activated B cells, T cells and myeloid cells (Agata et al.1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 havebeen shown to downregulate T cell activation upon binding to PD1(Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 NatImmunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 isabundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank etal. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 ClinCancer Res 10:5094). Immune suppression can be reversed by inhibitingthe local interaction of PD1 with PD-L1.

In one embodiment, the agent comprises the extracellular domain (ECD) ofan inhibitory molecule, e.g., Programmed Death 1 (PD1), can be fused toa transmembrane domain and intracellular signaling domains such as 41BBand CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment,the PD1 CAR, when used in combinations with an XCAR described herein,improves the persistence of the T cell. In one embodiment, the CAR is aPD1 CAR comprising the extracellular domain of PD1 indicated asunderlined in SEQ ID NO: 105. In one embodiment, the PD1 CAR comprisesthe amino acid sequence of SEQ ID NO:105.

(SEQ ID NO: 105) Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpsgqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgreeydvdldkrrgrdpemggkpffknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr.

In one embodiment, the PD1 CAR comprises the amino acid sequenceprovided below (SEQ ID NO:106).

(SEQ ID NO: 106) pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfylnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslraelryterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvillslvidycicrgrkkllyiflcqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr.

In one embodiment, the agent comprises a nucleic acid sequence encodingthe PD1 CAR, e.g., the PD1 CAR described herein. In one embodiment, thenucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECDunderlined below in SEQ ID NO: 107

(SEQ ID NO: 107) atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcactcttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaatcattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtttccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaatggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctgtgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactgagagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcctgcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggccccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccggaggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggaggaggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacgaactgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatacgatgccctgcacatgcaggcccttccccctcgc.

In another example, in one embodiment, the agent which enhances theactivity of a CAR-expressing cell can be a costimulatory molecule orcostimulatory molecule ligand. Examples of costimulatory moleculesinclude MHC class I molecule, BTLA and a Toll ligand receptor, as wellas OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and4-1BB (CD137). Further examples of such costimulatory molecules includeCDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44,NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2Rgamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6,CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b,ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C,TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a,and a ligand that specifically binds with CD83, e.g., as describedherein. Examples of costimulatory molecule ligands include CD80, CD86,CD40L, ICOSL, CD70, OX40L, 4-1BBL, GITRL, and LIGHT. In embodiments, thecostimulatory molecule ligand is a ligand for a costimulatory moleculedifferent from the costimulatory molecule domain of the CAR. Inembodiments, the costimulatory molecule ligand is a ligand for acostimulatory molecule that is the same as the costimulatory moleculedomain of the CAR. In an embodiment, the costimulatory molecule ligandis 4-1BBL. In an embodiment, the costimulatory ligand is CD80 or CD86.In an embodiment, the costimulatory molecule ligand is CD70. Inembodiments, a CAR-expressing immune effector cell described herein canbe further engineered to express one or more additional costimulatorymolecules or costimulatory molecule ligands.

Co-Expression of CAR with a Chemokine Receptor

In embodiments, the CAR-expressing cell described herein, e.g., CD19CAR-expressing cell, further comprises a chemokine receptor molecule.Transgenic expression of chemokine receptors CCR2b or CXCR2 in T cellsenhances trafficking to CCL2- or CXCL1-secreting solid tumors includingmelanoma and neuroblastoma (Craddock et al., J Immunother. 2010 October;33(8):780-8 and Kershaw et al., Hum Gene Ther. 2002 Nov. 1;13(16):1971-80). Thus, without wishing to be bound by theory, it isbelieved that chemokine receptors expressed in CAR-expressing cells thatrecognize chemokines secreted by tumors, e.g., solid tumors, can improvehoming of the CAR-expressing cell to the tumor, facilitate theinfiltration of the CAR-expressing cell to the tumor, and enhancesantitumor efficacy of the CAR-expressing cell. The chemokine receptormolecule can comprise a naturally occurring or recombinant chemokinereceptor or a chemokine-binding fragment thereof. A chemokine receptormolecule suitable for expression in a CAR-expressing cell (e.g., CAR-Tx)described herein include a CXC chemokine receptor (e.g., CXCR1, CXCR2,CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g.,CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11),a CX3C chemokine receptor (e.g., CX3CR1), a XC chemokine receptor (e.g.,XCR1), or a chemokine-binding fragment thereof. In one embodiment, thechemokine receptor molecule to be expressed with a CAR described hereinis selected based on the chemokine(s) secreted by the tumor. In oneembodiment, the CAR-expressing cell described herein further comprises,e.g., expresses, a CCR2b receptor or a CXCR2 receptor. In an embodiment,the CAR described herein and the chemokine receptor molecule are on thesame vector or are on two different vectors. In embodiments where theCAR described herein and the chemokine receptor molecule are on the samevector, the CAR and the chemokine receptor molecule are each undercontrol of two different promoters or are under the control of the samepromoter.

Nucleic Acid Constructs Encoding a CAR

The present invention also provides an immune effector cell, e.g., madeby a method described herein, that includes a nucleic acid moleculesencoding one or more CAR constructs described herein. In one aspect, thenucleic acid molecule is provided as a messenger RNA transcript. In oneaspect, the nucleic acid molecule is provided as a DNA construct.

The nucleic acid molecules described herein can be a DNA molecule, anRNA molecule, or a combination thereof. In one embodiment, the nucleicacid molecule is an mRNA encoding a CAR polypeptide as described herein.In other embodiments, the nucleic acid molecule is a vector thatincludes any of the aforesaid nucleic acid molecules.

In one aspect, the antigen binding domain of a CAR of the invention(e.g., a scFv) is encoded by a nucleic acid molecule whose sequence hasbeen codon optimized for expression in a mammalian cell. In one aspect,entire CAR construct of the invention is encoded by a nucleic acidmolecule whose entire sequence has been codon optimized for expressionin a mammalian cell. Codon optimization refers to the discovery that thefrequency of occurrence of synonymous codons (i.e., codons that code forthe same amino acid) in coding DNA is biased in different species. Suchcodon degeneracy allows an identical polypeptide to be encoded by avariety of nucleotide sequences. A variety of codon optimization methodsis known in the art, and include, e.g., methods disclosed in at leastU.S. Pat. Nos. 5,786,464 and 6,114,148.

Accordingly, in one aspect, an immune effector cell, e.g., made by amethod described herein, includes a nucleic acid molecule encoding achimeric antigen receptor (CAR), wherein the CAR comprises an antigenbinding domain that binds to a tumor antigen described herein, atransmembrane domain (e.g., a transmembrane domain described herein),and an intracellular signaling domain (e.g., an intracellular signalingdomain described herein) comprising a stimulatory domain, e.g., acostimulatory signaling domain (e.g., a costimulatory signaling domaindescribed herein) and/or a primary signaling domain (e.g., a primarysignaling domain described herein, e.g., a zeta chain described herein).

The present invention also provides vectors in which a nucleic acidmolecule encoding a CAR, e.g., a nucleic acid molecule described herein,is inserted. Vectors derived from retroviruses such as the lentivirusare suitable tools to achieve long-term gene transfer since they allowlong-term, stable integration of a transgene and its propagation indaughter cells. Lentiviral vectors have the added advantage over vectorsderived from onco-retroviruses such as murine leukemia viruses in thatthey can transduce non-proliferating cells, such as hepatocytes. Theyalso have the added advantage of low immunogenicity. A retroviral vectormay also be, e.g., a gammaretroviral vector. A gammaretroviral vectormay include, e.g., a promoter, a packaging signal (ψ), a primer bindingsite (PBS), one or more (e.g., two) long terminal repeats (LTR), and atransgene of interest, e.g., a gene encoding a CAR. A gammaretroviralvector may lack viral structural gens such as gag, pol, and env.Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV),Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus(MPSV), and vectors derived therefrom. Other gammaretroviral vectors aredescribed, e.g., in Tobias Maetzig et al., “Gammaretroviral Vectors:Biology, Technology and Application” Viruses. 2011 June; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encodingthe desired CAR is an adenoviral vector (A5/35). In another embodiment,the expression of nucleic acids encoding CARs can be accomplished usingof transposons such as sleeping beauty, crisper, CAS9, and zinc fingernucleases. See below June et al. 2009 Nature Reviews Immunology 9.10:704-716, is incorporated herein by reference.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration eukaryotes. Typical cloningvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al., 2012, MOLECULAR CLONING: ALABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave been shown to contain functional elements downstream of the startsite as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription. Exemplary promoters include theCMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK)promoters.

An example of a promoter that is capable of expressing a CAR encodingnucleic acid molecule in a mammalian T cell is the EF1a promoter. Thenative EF1a promoter drives expression of the alpha subunit of theelongation factor-1 complex, which is responsible for the enzymaticdelivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has beenextensively used in mammalian expression plasmids and has been shown tobe effective in driving CAR expression from nucleic acid moleculescloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther.17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises thesequence provided in the Examples.

Another example of a promoter is the immediate early cytomegalovirus(CMV) promoter sequence. This promoter sequence is a strong constitutivepromoter sequence capable of driving high levels of expression of anypolynucleotide sequence operatively linked thereto. However, otherconstitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, as well as human gene promoters such as, but not limitedto, the actin promoter, the myosin promoter, the elongation factor-1αpromoter, the hemoglobin promoter, and the creatine kinase promoter.Further, the invention should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of theinvention. The use of an inducible promoter provides a molecular switchcapable of turning on expression of the polynucleotide sequence which itis operatively linked when such expression is desired, or turning offthe expression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

Another example of a promoter is the phosphoglycerate kinase (PGK)promoter. In embodiments, a truncated PGK promoter (e.g., a PGK promoterwith one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotidedeletions when compared to the wild-type PGK promoter sequence) may bedesired.

The nucleotide sequences of exemplary PGK promoters are provided below.

WT PGK Promoter: (SEQ ID NO: 109)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGGTGCGGGTCTCGTCGGCGCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGCGTTGGGGTTGGGGCACCATAAGCT  Exemplary truncated PGK Promoters: PGK100:(SEQ ID NO: 110) ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCC GGGTGTGATGGCGGGGTG PGK200: (SEQ ID NO: 111)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACG  PGK300: (SEQ ID NO: 112)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCG  PGK400: (SEQ ID NO: 113)ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGGCGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACCGGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACGCTCCCATGATCACTCTGCACGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCTGAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACTGCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCG 

A vector may also include, e.g., a signal sequence to facilitatesecretion, a polyadenylation signal and transcription terminator (e.g.,from Bovine Growth Hormone (BGH) gene), an element allowing episomalreplication and replication in prokaryotes (e.g. SV40 origin and ColE1or others known in the art) and/or elements to allow selection (e.g.,ampicillin resistance gene and/or zeocin marker).

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In embodiments, the vector may comprise two or more nucleic acidsequences encoding a CAR, e.g., a CAR described herein, e.g., a CD19CAR, and a second CAR, e.g., an inhibitory CAR or a CAR thatspecifically binds to an antigen other than CD19. In such embodiments,the two or more nucleic acid sequences encoding the CAR are encoded by asingle nucleic molecule in the same frame and as a single polypeptidechain. In this aspect, the two or more CARs, can, e.g., be separated byone or more peptide cleavage sites. (e.g., an auto-cleavage site or asubstrate for an intracellular protease). Examples of peptide cleavagesites include T2A, P2A, E2A, or F2A sites.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method, e.g., one known in the art. For example, theexpression vector can be transferred into a host cell by physical,chemical, or biological means.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al., 2012,MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring HarborPress, NY). A suitable method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). Other methodsof state-of-the-art targeted delivery of nucleic acids are available,such as delivery of polynucleotides with targeted nanoparticles or othersuitable sub-micron sized delivery system.

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant nucleicacid sequence in the host cell, a variety of assays may be performed.Such assays include, for example, “molecular biological” assays wellknown to those of skill in the art, such as Southern and Northernblotting, RT-PCR and PCR; “biochemical” assays, such as detecting thepresence or absence of a particular peptide, e.g., by immunologicalmeans (ELISAs and Western blots) or by assays described herein toidentify agents falling within the scope of the invention.

Natural Killer Cell Receptor (NKR) CARs

In an embodiment, the CAR molecule described herein comprises one ormore components of a natural killer cell receptor (NKR), thereby formingan NKR-CAR. The NKR component can be a transmembrane domain, a hingedomain, or a cytoplasmic domain from any of the following natural killercell receptors: killer cell immunoglobulin-like receptor (KIR), e.g.,KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2,KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, andKIR3DP1; natural cytotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46;signaling lymphocyte activation molecule (SLAM) family of immune cellreceptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, andCD2F-10; Fc receptor (FcR), e.g., CD16, and CD64; and Ly49 receptors,e.g., LY49A, LY49C. The NKR-CAR molecules described herein may interactwith an adaptor molecule or intracellular signaling domain, e.g., DAP12.Exemplary configurations and sequences of CAR molecules comprising NKRcomponents are described in International Publication No. WO2014/145252,the contents of which are hereby incorporated by reference.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The splitCAR approach is described in more detail in publications WO2014/055442and WO2014/055657. Briefly, a split CAR system comprises a cellexpressing a first CAR having a first antigen binding domain and acostimulatory domain (e.g., 41BB), and the cell also expresses a secondCAR having a second antigen binding domain and an intracellularsignaling domain (e.g., CD3 zeta). When the cell encounters the firstantigen, the costimulatory domain is activated, and the cellproliferates. When the cell encounters the second antigen, theintracellular signaling domain is activated and cell-killing activitybegins. Thus, the CAR-expressing cell is only fully activated in thepresence of both antigens.

Strategies for Regulating Chimeric Antigen Receptors

In some embodiments, a regulatable CAR (RCAR) where the CAR activity canbe controlled is desirable to optimize the safety and efficacy of a CARtherapy. There are many ways CAR activities can be regulated. Forexample, inducible apoptosis using, e.g., a caspase fused to adimerization domain (see, e.g., Di Stasa et al., N Engl. J. Med. 2011Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CARtherapy of the instant invention. In one embodiment, the cells (e.g., Tcells or NK cells) expressing a CAR of the present invention furthercomprise an inducible apoptosis switch, wherein a human caspase (e.g.,caspase 9) or a modified version is fused to a modification of the humanFKB protein that allows conditional dimerization. In the presence of asmall molecule, such as a rapalog (e.g., AP 1903, AP20187), theinducible caspase (e.g., caspase 9) is activated and leads to the rapidapoptosis and death of the cells (e.g., T cells or NK cells) expressinga CAR of the present invention. Examples of a caspase-based inducibleapoptosis switch (or one or more aspects of such a switch) have beendescribed in, e.g., US2004040047; US20110286980; US20140255360;WO1997031899; WO2014151960; WO2014164348; WO2014197638; WO2014197638;all of which are incorporated by reference herein.

In another example, CAR-expressing cells can also express an inducibleCaspase-9 (iCaspase-9) molecule that, upon administration of a dimerizerdrug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) orAP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis ofthe cells. The iCaspase-9 molecule contains a chemical inducer ofdimerization (CID) binding domain that mediates dimerization in thepresence of a CID. This results in inducible and selective depletion ofCAR-expressing cells. In some cases, the iCaspase-9 molecule is encodedby a nucleic acid molecule separate from the CAR-encoding vector(s). Insome cases, the iCaspase-9 molecule is encoded by the same nucleic acidmolecule as the CAR-encoding vector. The iCaspase-9 can provide a safetyswitch to avoid any toxicity of CAR-expressing cells. See, e.g., Song etal. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No.NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365:1673-83.

Alternative strategies for regulating the CAR therapy of the instantinvention include utilizing small molecules or antibodies thatdeactivate or turn off CAR activity, e.g., by deleting CAR-expressingcells, e.g., by inducing antibody dependent cell-mediated cytotoxicity(ADCC). For example, CAR-expressing cells described herein may alsoexpress an antigen that is recognized by molecules capable of inducingcell death, e.g., ADCC or complement-induced cell death. For example,CAR expressing cells described herein may also express a receptorcapable of being targeted by an antibody or antibody fragment. Examplesof such receptors include EpCAM, VEGFR, integrins (e.g., integrins αvβ3,α4, αI3/4β3, α4β7, α5β1, αvβ3, αv), members of the TNF receptorsuperfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferonreceptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1,TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/IgE Receptor, CD25,CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80,CD125, CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5,CD319/SLAMF7, and EGFR, and truncated versions thereof (e.g., versionspreserving one or more extracellular epitopes but lacking one or moreregions within the cytoplasmic domain).

For example, a CAR-expressing cell described herein may also express atruncated epidermal growth factor receptor (EGFR) which lacks signalingcapacity but retains the epitope that is recognized by molecules capableof inducing ADCC, e.g., cetuximab (ERBITUX®), such that administrationof cetuximab induces ADCC and subsequent depletion of the CAR-expressingcells (see, e.g., WO2011/056894, and Jonnalagadda et al., Gene Ther.2013; 20(8)853-860). Another strategy includes expressing a highlycompact marker/suicide gene that combines target epitopes from both CD32and CD20 antigens in the CAR-expressing cells described herein, whichbinds rituximab, resulting in selective depletion of the CAR-expressingcells, e.g., by ADCC (see, e.g., Philip et al., Blood. 2014;124(8)1277-1287). Other methods for depleting CAR-expressing cellsdescribed herein include administration of CAMPATH, a monoclonalanti-CD52 antibody that selectively binds and targets maturelymphocytes, e.g., CAR-expressing cells, for destruction, e.g., byinducing ADCC. In other embodiments, the CAR-expressing cell can beselectively targeted using a CAR ligand, e.g., an anti-idiotypicantibody. In some embodiments, the anti-idiotypic antibody can causeeffector cell activity, e.g., ADCC or ADC activities, thereby reducingthe number of CAR-expressing cells. In other embodiments, the CARligand, e.g., the anti-idiotypic antibody, can be coupled to an agentthat induces cell killing, e.g., a toxin, thereby reducing the number ofCAR-expressing cells. Alternatively, the CAR molecules themselves can beconfigured such that the activity can be regulated, e.g., turned on andoff, as described below.

In other embodiments, a CAR-expressing cell described herein may alsoexpress a target protein recognized by the T cell depleting agent. Inone embodiment, the target protein is CD20 and the T cell depletingagent is an anti-CD20 antibody, e.g., rituximab. In such embodiment, theT cell depleting agent is administered once it is desirable to reduce oreliminate the CAR-expressing cell, e.g., to mitigate the CAR inducedtoxicity. In other embodiments, the T cell depleting agent is ananti-CD52 antibody, e.g., alemtuzumab, as described in the Examplesherein.

In other embodiments, an RCAR comprises a set of polypeptides, typicallytwo in the simplest embodiments, in which the components of a standardCAR described herein, e.g., an antigen binding domain and anintracellular signalling domain, are partitioned on separatepolypeptides or members. In some embodiments, the set of polypeptidesinclude a dimerization switch that, upon the presence of a dimerizationmolecule, can couple the polypeptides to one another, e.g., can couplean antigen binding domain to an intracellular signalling domain. In oneembodiment, a CAR of the present invention utilizes a dimerizationswitch as those described in, e.g., WO2014127261, which is incorporatedby reference herein. Additional description and exemplary configurationsof such regulatable CARs are provided herein and in, e.g., paragraphs527-551 of International Publication No. WO 2015/090229 filed Mar. 13,2015, which is incorporated by reference in its entirety. In someembodiments, an RCAR involves a switch domain, e.g., a FKBP switchdomain, as set out SEQ ID NO: 114, or comprise a fragment of FKBP havingthe ability to bind with FRB, e.g., as set out in SEQ ID NO: 115. Insome embodiments, the RCAR involves a switch domain comprising a FRBsequence, e.g., as set out in SEQ ID NO: 116, or a mutant FRB sequence,e.g., as set out in any of SEQ ID Nos. 117-122.

(SEQ ID NO: 114) DVPDYASLGGPSSPKKKRKVSRGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLETSY  (SEQ ID NO: 115)VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV  ELLKLETS(SEQ ID NO: 116) ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK 

TABLE 4Exemplary mutant FRB having increased affinity for a dimerization molecule.SEQ ID FRB mutant Amino Acid Sequence NO: E20321 mutantILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG 117RDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS E2032L mutantILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG 118RDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS T2098L mutantILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG 119RDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032, T2098 ILWHEMWHEGL XEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG 120 mutantRDLMEAQEWCRKYMKSGNVKDL X QAWDLYYHVFRRISKTS E2032I, T2098LILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG 121 mutantRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS E2032L, T2098LILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG 122 mutantRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTSRNA Transfection

Disclosed herein are methods for producing an in vitro transcribed RNACAR. RNA CAR and methods of using the same are described, e.g., inparagraphs 553-570 of in International Application WO2015/142675, filedMar. 13, 2015, which is herein incorporated by reference in itsentirety.

An immune effector cell can include a CAR encoded by a messenger RNA(mRNA). In one aspect, the mRNA encoding a CAR described herein isintroduced into an immune effector cell, e.g., made by a methoddescribed herein, for production of a CAR-expressing cell.

In one embodiment, the in vitro transcribed RNA CAR can be introduced toa cell as a form of transient transfection. The RNA is produced by invitro transcription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired temple for in vitrotranscription is a CAR described herein. For example, the template forthe RNA CAR comprises an extracellular region comprising a single chainvariable domain of an antibody to a tumor associated antigen describedherein; a hinge region (e.g., a hinge region described herein), atransmembrane domain (e.g., a transmembrane domain described herein suchas a transmembrane domain of CD8a); and a cytoplasmic region thatincludes an intracellular signaling domain, e.g., an intracellularsignaling domain described herein, e.g., comprising the signaling domainof CD3-zeta and the signaling domain of 4-1BB.

In one embodiment, the DNA to be used for PCR contains an open readingframe. The DNA can be from a naturally occurring DNA sequence from thegenome of an organism. In one embodiment, the nucleic acid can includesome or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleicacid can include exons and introns. In one embodiment, the DNA to beused for PCR is a human nucleic acid sequence. In another embodiment,the DNA to be used for PCR is a human nucleic acid sequence includingthe 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNAsequence that is not normally expressed in a naturally occurringorganism. An exemplary artificial DNA sequence is one that containsportions of genes that are ligated together to form an open readingframe that encodes a fusion protein. The portions of DNA that areligated together can be from a single organism or from more than oneorganism.

PCR is used to generate a template for in vitro transcription of mRNAwhich is used for transfection. Methods for performing PCR are wellknown in the art. Primers for use in PCR are designed to have regionsthat are substantially complementary to regions of the DNA to be used asa template for the PCR. “Substantially complementary,” as used herein,refers to sequences of nucleotides where a majority or all of the basesin the primer sequence are complementary, or one or more bases arenon-complementary, or mismatched. Substantially complementary sequencesare able to anneal or hybridize with the intended DNA target underannealing conditions used for PCR. The primers can be designed to besubstantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a nucleicacid that is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a nucleic acid that encodes a particular domain of interest.In one embodiment, the primers are designed to amplify the coding regionof a human cDNA, including all or portions of the 5′ and 3′ UTRs.Primers useful for PCR can be generated by synthetic methods that arewell known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Any DNA polymerase useful for PCR can be used in the methods disclosedherein. The reagents and polymerase are commercially available from anumber of sources.

Chemical structures with the ability to promote stability and/ortranslation efficiency may also be used. The RNA in embodiments has 5′and 3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000nucleotides in length. The length of 5′ and 3′ UTR sequences to be addedto the coding region can be altered by different methods, including, butnot limited to, designing primers for PCR that anneal to differentregions of the UTRs. Using this approach, one of ordinary skill in theart can modify the 5′ and 3′ UTR lengths required to achieve optimaltranslation efficiency following transfection of the transcribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the nucleic acid of interest. Alternatively, UTR sequences thatare not endogenous to the nucleic acid of interest can be added byincorporating the UTR sequences into the forward and reverse primers orby any other modifications of the template. The use of UTR sequencesthat are not endogenous to the nucleic acid of interest can be usefulfor modifying the stability and/or translation efficiency of the RNA.For example, it is known that AU-rich elements in 3′ UTR sequences candecrease the stability of mRNA. Therefore, 3′ UTRs can be selected ordesigned to increase the stability of the transcribed RNA based onproperties of UTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous nucleic acid. Alternatively, when a 5′ UTR that is notendogenous to the nucleic acid of interest is being added by PCR asdescribed above, a consensus Kozak sequence can be redesigned by addingthe 5′ UTR sequence. Kozak sequences can increase the efficiency oftranslation of some RNA transcripts, but does not appear to be requiredfor all RNAs to enable efficient translation. The requirement for Kozaksequences for many mRNAs is known in the art. In other embodiments the5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells.In other embodiments various nucleotide analogues can be used in the 3′or 5′ UTR to impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In an embodiment, the mRNA has both a cap on the 5′ end and a 3′ poly(A)tail which determine ribosome binding, initiation of translation andstability mRNA in the cell. On a circular DNA template, for instance,plasmid DNA, RNA polymerase produces a long concatameric product whichis not suitable for expression in eukaryotic cells. The transcription ofplasmid DNA linearized at the end of the 3′ UTR results in normal sizedmRNA which is not effective in eukaryotic transfection even if it ispolyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (SEQ ID NO: 31) (size can be 50-5000 T (SEQ ID NO: 32)), orafter PCR by any other method, including, but not limited to, DNAligation or in vitro recombination. Poly(A) tails also provide stabilityto RNAs and reduce their degradation. Generally, the length of a poly(A)tail positively correlates with the stability of the transcribed RNA. Inone embodiment, the poly(A) tail is between 100 and 5000 adenosines(e.g., SEQ ID NO: 33).

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotides(SEQ ID NO: 34) results in about a two-fold increase in the translationefficiency of the RNA. Additionally, the attachment of differentchemical groups to the 3′ end can increase mRNA stability. Suchattachment can contain modified/artificial nucleotides, aptamers andother compounds. For example, ATP analogs can be incorporated into thepoly(A) tail using poly(A) polymerase. ATP analogs can further increasethe stability of the RNA.

5′ caps on also provide stability to RNA molecules. In an embodiment,RNAs produced by the methods disclosed herein include a 5′ cap. The 5′cap is provided using techniques known in the art and described herein(Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski,et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res.Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Non-Viral Delivery Methods

In some aspects, non-viral methods can be used to deliver a nucleic acidencoding a CAR described herein into a cell or tissue or a subject.

In some embodiments, the non-viral method includes the use of atransposon (also called a transposable element). In some embodiments, atransposon is a piece of DNA that can insert itself at a location in agenome, for example, a piece of DNA that is capable of self-replicatingand inserting its copy into a genome, or a piece of DNA that can bespliced out of a longer nucleic acid and inserted into another place ina genome. For example, a transposon comprises a DNA sequence made up ofinverted repeats flanking genes for transposition.

Exemplary methods of nucleic acid delivery using a transposon include aSleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposonsystem. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al.Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther.18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams.Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protoc.2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83, all ofwhich are incorporated herein by reference.

The SBTS includes two components: 1) a transposon containing a transgeneand 2) a source of transposase enzyme. The transposase can transpose thetransposon from a carrier plasmid (or other donor DNA) to a target DNA,such as a host cell chromosome/genome. For example, the transposasebinds to the carrier plasmid/donor DNA, cuts the transposon (includingtransgene(s)) out of the plasmid, and inserts it into the genome of thehost cell. See, e.g., Aronovich et al. supra.

Exemplary transposons include a pT2-based transposon. See, e.g.,Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh etal. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporatedherein by reference. Exemplary transposases include a Tc1/mariner-typetransposase, e.g., the SB10 transposase or the SB11 transposase (ahyperactive transposase which can be expressed, e.g., from acytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.;and Grabundzija et al., all of which are incorporated herein byreference.

Use of the SBTS permits efficient integration and expression of atransgene, e.g., a nucleic acid encoding a CAR described herein.Provided herein are methods of generating a cell, e.g., T cell or NKcell, that stably expresses a CAR described herein, e.g., using atransposon system such as SBTS.

In accordance with methods described herein, in some embodiments, one ormore nucleic acids, e.g., plasmids, containing the SBTS components aredelivered to a cell (e.g., T or NK cell). For example, the nucleicacid(s) are delivered by standard methods of nucleic acid (e.g., plasmidDNA) delivery, e.g., methods described herein, e.g., electroporation,transfection, or lipofection. In some embodiments, the nucleic acidcontains a transposon comprising a transgene, e.g., a nucleic acidencoding a CAR described herein. In some embodiments, the nucleic acidcontains a transposon comprising a transgene (e.g., a nucleic acidencoding a CAR described herein) as well as a nucleic acid sequenceencoding a transposase enzyme. In other embodiments, a system with twonucleic acids is provided, e.g., a dual-plasmid system, e.g., where afirst plasmid contains a transposon comprising a transgene, and a secondplasmid contains a nucleic acid sequence encoding a transposase enzyme.For example, the first and the second nucleic acids are co-deliveredinto a host cell.

In some embodiments, cells, e.g., T or NK cells, are generated thatexpress a CAR described herein by using a combination of gene insertionusing the SBTS and genetic editing using a nuclease (e.g., Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, or engineered meganucleasere-engineered homing endonucleases).

In some embodiments, use of a non-viral method of delivery permitsreprogramming of cells, e.g., T or NK cells, and direct infusion of thecells into a subject. Advantages of non-viral vectors include but arenot limited to the ease and relatively low cost of producing sufficientamounts required to meet a patient population, stability during storage,and lack of immunogenicity.

Methods of Manufacture/Production

In some embodiments, the methods disclosed herein further includeadministering a T cell depleting agent after treatment with the cell(e.g., an immune effector cell as described herein), thereby reducing(e.g., depleting) the CAR-expressing cells (e.g., the CD19CAR-expressingcells). Such T cell depleting agents can be used to effectively depleteCAR-expressing cells (e.g., CD19CAR-expressing cells) to mitigatetoxicity. In some embodiments, the CAR-expressing cells weremanufactured according to a method herein, e.g., assayed (e.g., beforeor after transfection or transduction) according to a method herein.

In some embodiments, the T cell depleting agent is administered one,two, three, four, or five weeks after administration of the cell, e.g.,the population of immune effector cells, described herein.

In one embodiment, the T cell depleting agent is an agent that depletesCAR-expressing cells, e.g., by inducing antibody dependent cell-mediatedcytotoxicity (ADCC) and/or complement-induced cell death. For example,CAR-expressing cells described herein may also express an antigen (e.g.,a target antigen) that is recognized by molecules capable of inducingcell death, e.g., ADCC or complement-induced cell death. For example,CAR expressing cells described herein may also express a target protein(e.g., a receptor) capable of being targeted by an antibody or antibodyfragment. Examples of such target proteins include, but are not limitedto, EpCAM, VEGFR, integrins (e.g., integrins αvβ3, α4, αI3/4β3, α4β7,α5β1, αvβ3, αv), members of the TNF receptor superfamily (e.g.,TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon receptor, folatereceptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15,CD18/ITGB2, CD19, CD20, CD22, CD23/IgE Receptor, CD25, CD28, CD30, CD33,CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125,CD147/basigin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, andEGFR, and truncated versions thereof (e.g., versions preserving one ormore extracellular epitopes but lacking one or more regions within thecytoplasmic domain).

In some embodiments, the CAR expressing cell co-expresses the CAR andthe target protein, e.g., naturally expresses the target protein or isengineered to express the target protein. For example, the cell, e.g.,the population of immune effector cells, can include a nucleic acid(e.g., vector) comprising the CAR nucleic acid (e.g., a CAR nucleic acidas described herein) and a nucleic acid encoding the target protein.

In one embodiment, the T cell depleting agent is a CD52 inhibitor, e.g.,an anti-CD52 antibody molecule, e.g., alemtuzumab.

In other embodiments, the cell, e.g., the population of immune effectorcells, expresses a CAR molecule as described herein (e.g., CD19CAR) andthe target protein recognized by the T cell depleting agent. In oneembodiment, the target protein is CD20. In embodiments where the targetprotein is CD20, the T cell depleting agent is an anti-CD20 antibody,e.g., rituximab.

In further embodiments of any of the aforesaid methods, the methodsfurther include transplanting a cell, e.g., a hematopoietic stem cell,or a bone marrow, into the mammal.

In another aspect, the invention features a method of conditioning amammal prior to cell transplantation. The method includes administeringto the mammal an effective amount of the cell comprising a CAR nucleicacid or polypeptide, e.g., a CD19 CAR nucleic acid or polypeptide. Insome embodiments, the cell transplantation is a stem celltransplantation, e.g., a hematopoietic stem cell transplantation, or abone marrow transplantation. In other embodiments, conditioning asubject prior to cell transplantation includes reducing the number oftarget-expressing cells in a subject, e.g., CD19-expressing normal cellsor CD19-expressing cancer cells.

Activation and Expansion of Immune Effector Cells (e.g., T Cells)

Immune effector cells such as T cells may be activated and expandedgenerally using methods as described, for example, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005.

Generally, a population of immune effector cells, e.g., T regulatorycell depleted cells, may be expanded by contact with a surface havingattached thereto an agent that stimulates a CD3/TCR complex associatedsignal and a ligand that stimulates a costimulatory molecule on thesurface of the T cells. In particular, T cell populations may bestimulated as described herein, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4+T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibodycan be used. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain aspects, the primary stimulatory signal and the costimulatorysignal for the T cell may be provided by different protocols. Forexample, the agents providing each signal may be in solution or coupledto a surface. When coupled to a surface, the agents may be coupled tothe same surface (i.e., in “cis” formation) or to separate surfaces(i.e., in “trans” formation). Alternatively, one agent may be coupled toa surface and the other agent in solution. In one aspect, the agentproviding the costimulatory signal is bound to a cell surface and theagent providing the primary activation signal is in solution or coupledto a surface. In certain aspects, both agents can be in solution. In oneaspect, the agents may be in soluble form, and then cross-linked to asurface, such as a cell expressing Fc receptors or an antibody or otherbinding agent which will bind to the agents. In this regard, see forexample, U.S. Patent Application Publication Nos. 20040101519 and20060034810 for artificial antigen presenting cells (aAPCs) that arecontemplated for use in activating and expanding T cells in the presentinvention.

In one aspect, the two agents are immobilized on beads, either on thesame bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way ofexample, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the costimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one aspect, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects of the present invention, aratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular aspect an increase offrom about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one aspect, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect, more anti-CD28 antibody is bound tothe particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 isless than one. In certain aspects, the ratio of anti CD28 antibody toanti CD3 antibody bound to the beads is greater than 2:1. In oneparticular aspect, a 1:100 CD3:CD28 ratio of antibody bound to beads isused. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to beads isused. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound tobeads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound tobeads is used. In one aspect, a 1:10 CD3:CD28 ratio of antibody bound tobeads is used. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound tothe beads is used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibodybound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain aspects the ratio of cells toparticles ranges from 1:100 to 100:1 and any integer values in-betweenand in further aspects the ratio comprises 1:9 to 9:1 and any integervalues in between, can also be used to stimulate T cells. The ratio ofanti-CD3- and anti-CD28-coupled particles to T cells that result in Tcell stimulation can vary as noted above, however certain suitablevalues include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6,1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,and 15:1 with one suitable ratio being at least 1:1 particles per Tcell. In one aspect, a ratio of particles to cells of 1:1 or less isused. In one particular aspect, a suitable particle: cell ratio is 1:5.In further aspects, the ratio of particles to cells can be varieddepending on the day of stimulation. For example, in one aspect, theratio of particles to cells is from 1:1 to 10:1 on the first day andadditional particles are added to the cells every day or every other daythereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (basedon cell counts on the day of addition). In one particular aspect, theratio of particles to cells is 1:1 on the first day of stimulation andadjusted to 1:5 on the third and fifth days of stimulation. In oneaspect, particles are added on a daily or every other day basis to afinal ratio of 1:1 on the first day, and 1:5 on the third and fifth daysof stimulation. In one aspect, the ratio of particles to cells is 2:1 onthe first day of stimulation and adjusted to 1:10 on the third and fifthdays of stimulation. In one aspect, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type. In one aspect, the most typical ratiosfor use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.

In further aspects, the cells, such as T cells, are combined withagent-coated beads, the beads and the cells are subsequently separated,and then the cells are cultured. In an alternative aspect, prior toculture, the agent-coated beads and cells are not separated but arecultured together. In a further aspect, the beads and cells are firstconcentrated by application of a force, such as a magnetic force,resulting in increased ligation of cell surface markers, therebyinducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one aspect the cells (for example, 10⁴to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1) are combined in a buffer, forexample PBS (without divalent cations such as, calcium and magnesium).Again, those of ordinary skill in the art can readily appreciate anycell concentration may be used. For example, the target cell may be veryrare in the sample and comprise only 0.01% of the sample or the entiresample (i.e., 100%) may comprise the target cell of interest.Accordingly, any cell number is within the context of the presentinvention. In certain aspects, it may be desirable to significantlydecrease the volume in which particles and cells are mixed together(i.e., increase the concentration of cells), to ensure maximum contactof cells and particles. For example, in one aspect, a concentration ofabout 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect,greater than 100 million cells/ml is used. In a further aspect, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet one aspect, a concentration of cells from 75,80, 85, 90, 95, or 100 million cells/ml is used. In further aspects,concentrations of 125 or 150 million cells/ml can be used. Using highconcentrations can result in increased cell yield, cell activation, andcell expansion. Further, use of high cell concentrations allows moreefficient capture of cells that may weakly express target antigens ofinterest, such as CD28-negative T cells. Such populations of cells mayhave therapeutic value and would be desirable to obtain in certainaspects. For example, using high concentration of cells allows moreefficient selection of CD8+ T cells that normally have weaker CD28expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR,e.g., a CAR described herein, e.g., a CD19 CAR described herein, areexpanded, e.g., by a method described herein. In one embodiment, thecells are expanded in culture for a period of several hours (e.g., about2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment,the cells are expanded for a period of 4 to 9 days. In one embodiment,the cells are expanded for a period of 8 days or less, e.g., 7, 6 or 5days. In one embodiment, the cells are expanded in culture for 5 days,and the resulting cells are more potent than the same cells expanded inculture for 9 days under the same culture conditions. Potency can bedefined, e.g., by various T cell functions, e.g. proliferation, targetcell killing, cytokine production, activation, migration, orcombinations thereof. In one embodiment, the cells, e.g., a CD19 CARcell described herein, expanded for 5 days show at least a one, two,three or four fold increase in cells doublings upon antigen stimulationas compared to the same cells expanded in culture for 9 days under thesame culture conditions. In one embodiment, the cells, e.g., the cellsexpressing a CD19 CAR described herein, are expanded in culture for 5days, and the resulting cells exhibit higher proinflammatory cytokineproduction, e.g., IFN-γ and/or GM-CSF levels, as compared to the samecells expanded in culture for 9 days under the same culture conditions.In one embodiment, the cells, e.g., a CD19 CAR cell described herein,expanded for 5 days show at least a one, two, three, four, five, tenfold or more increase in pg/ml of proinflammatory cytokine production,e.g., IFN-γ and/or GM-CSF levels, as compared to the same cells expandedin culture for 9 days under the same culture conditions.

Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any otheradditives for the growth of cells known to the skilled artisan. Otheradditives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplementedwith an appropriate amount of serum (or plasma) or a defined set ofhormones, and/or an amount of cytokine(s) sufficient for the growth andexpansion of T cells. Antibiotics, e.g., penicillin and streptomycin,are included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject. The target cells are maintainedunder conditions necessary to support growth, for example, anappropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5%CO₂).

In one embodiment, the cells are expanded in an appropriate media (e.g.,media described herein) that includes one or more interleukin thatresult in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold,350-fold) increase in cells over a 14 day expansion period, e.g., asmeasured by a method described herein such as flow cytometry. In oneembodiment, the cells are expanded in the presence IL-15 and/or IL-7(e.g., IL-15 and IL-7).

In embodiments, methods described herein, e.g., CAR-expressing cellmanufacturing methods, comprise removing T regulatory cells, e.g., CD25+T cells, from a cell population, e.g., using an anti-CD25 antibody, orfragment thereof, or a CD25-binding ligand, IL-2. Methods of removing Tregulatory cells, e.g., CD25+ T cells, from a cell population aredescribed herein. In embodiments, the methods, e.g., manufacturingmethods, further comprise contacting a cell population (e.g., a cellpopulation in which T regulatory cells, such as CD25+ T cells, have beendepleted; or a cell population that has previously contacted ananti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15and/or IL-7. For example, the cell population (e.g., that has previouslycontacted an anti-CD25 antibody, fragment thereof, or CD25-bindingligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contactedwith a composition comprising a interleukin-15 (IL-15) polypeptide, ainterleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination ofboth a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15,during the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising a IL-15 polypeptide during the manufacturing ofthe CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressingcell described herein is contacted with a composition comprising acombination of both a IL-15 polypeptide and a IL-15 Ra polypeptideduring the manufacturing of the CAR-expressing cell, e.g., ex vivo. Inembodiments, a CAR-expressing cell described herein is contacted with acomposition comprising hetIL-15 during the manufacturing of theCAR-expressing cell, e.g., ex vivo.

In one embodiment the CAR-expressing cell described herein is contactedwith a composition comprising hetIL-15 during ex vivo expansion. In anembodiment, the CAR-expressing cell described herein is contacted with acomposition comprising an IL-15 polypeptide during ex vivo expansion. Inan embodiment, the CAR-expressing cell described herein is contactedwith a composition comprising both an IL-15 polypeptide and an IL-15Rapolypeptide during ex vivo expansion. In one embodiment the contactingresults in the survival and proliferation of a lymphocyte subpopulation,e.g., CD8+ T cells.

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (TH, CD4+) that is greater than the cytotoxic or suppressor Tcell population (TC, CD8+). Ex vivo expansion of T cells by stimulatingCD3 and CD28 receptors produces a population of T cells that prior toabout days 8-9 consists predominately of TH cells, while after aboutdays 8-9, the population of T cells comprises an increasingly greaterpopulation of TC cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of TH cells may be advantageous. Similarly, if anantigen-specific subset of TC cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Once a CAR described herein is constructed, various assays can be usedto evaluate the activity of the molecule, such as but not limited to,the ability to expand T cells following antigen stimulation, sustain Tcell expansion in the absence of re-stimulation, and anti-canceractivities in appropriate in vitro and animal models. Assays to evaluatethe effects of a CAR of the present invention are described in furtherdetail below

Western blot analysis of CAR expression in primary T cells can be usedto detect the presence of monomers and dimers, e.g., as described inparagraph 695 of International Application WO2015/142675, filed Mar. 13,2015, which is herein incorporated by reference in its entirety.

In vitro expansion of CAR⁺ T cells following antigen stimulation can bemeasured by flow cytometry. For example, a mixture of CD4⁺ and CD8⁺ Tcells are stimulated with αCD3/αCD28 aAPCs followed by transduction withlentiviral vectors expressing GFP under the control of the promoters tobe analyzed. Exemplary promoters include the CMV IE gene, EF-1α,ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescenceis evaluated on day 6 of culture in the CD4⁺ and/or CD8⁺ T cell subsetsby flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Alternatively, a mixture of CD4⁺ and CD8⁺ T cells arestimulated with αCD3/αCD28 coated magnetic beads on day 0, andtransduced with CAR on day 1 using a bicistronic lentiviral vectorexpressing CAR along with eGFP using a 2A ribosomal skipping sequence.Cultures are re-stimulated with either a cancer associated antigen asdescribed herein⁺ K562 cells (K562-expressing a cancer associatedantigen as described herein), wild-type K562 cells (K562 wild type) orK562 cells expressing hCD32 and 4-1BBL in the presence of antiCD3 andanti-CD28 antibody (K562-BBL-3/28) following washing. Exogenous IL-2 isadded to the cultures every other day at 100 IU/ml. GFP⁺ T cells areenumerated by flow cytometry using bead-based counting. See, e.g.,Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

Sustained CAR⁺ T cell expansion in the absence of re-stimulation canalso be measured. See, e.g., Milone et al., Molecular Therapy 17(8):1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8of culture using a Coulter Multisizer III particle counter, a NexcelomCellometer Vision or Millipore Scepter, following stimulation withαCD3/αCD28 coated magnetic beads on day 0, and transduction with theindicated CAR on day 1.

Animal models can also be used to measure a CAR-expressing cellactivity, e.g., as described in paragraph 698 of InternationalApplication WO2015/142675, filed Mar. 13, 2015, which is hereinincorporated by reference in its entirety.

Dose dependent CAR treatment response can be evaluated, e.g., asdescribed in paragraph 699 of International Application WO2015/142675,filed Mar. 13, 2015, which is herein incorporated by reference in itsentirety.

Assessment of cell proliferation and cytokine production has beenpreviously described, as described in paragraph 700 of InternationalApplication WO2015/142675, filed Mar. 13, 2015, which is hereinincorporated by reference in its entirety.

Cytotoxicity can be assessed by a standard 51Cr-release assay, e.g., asdescribed in paragraph 701 of International Application WO2015/142675,filed Mar. 13, 2015, which is herein incorporated by reference in itsentirety.

Cytotoxicity can also be assessed by measuring changes in adherentcell's electrical impedance, e.g., using an xCELLigence real time cellanalyzer (RTCA). In some embodiments, cytotoxicity is measured atmultiple time points.

Imaging technologies can be used to evaluate specific trafficking andproliferation of CARs in tumor-bearing animal models, e.g., as describedin paragraph 702 of International Application WO2015/142675, filed Mar.13, 2015, which is herein incorporated by reference in its entirety.

Other assays, including those described in the Example section herein aswell as those that are known in the art can also be used to evaluate theCARs described herein.

Alternatively, or in combination to the methods disclosed herein,methods and compositions for one or more of: detection and/orquantification of CAR-expressing cells (e.g., in vitro or in vivo (e.g.,clinical monitoring)); immune cell expansion and/or activation; and/orCAR-specific selection, that involve the use of a CAR ligand, aredisclosed. In one exemplary embodiment, the CAR ligand is an antibodythat binds to the CAR molecule, e.g., binds to the extracellular antigenbinding domain of CAR (e.g., an antibody that binds to the antigenbinding domain, e.g., an anti-idiotypic antibody; or an antibody thatbinds to a constant region of the extracellular binding domain). Inother embodiments, the CAR ligand is a CAR antigen molecule (e.g., a CARantigen molecule as described herein).

In one aspect, a method for detecting and/or quantifying CAR-expressingcells is disclosed. For example, the CAR ligand can be used to detectand/or quantify CAR-expressing cells in vitro or in vivo (e.g., clinicalmonitoring of CAR-expressing cells in a patient, or dosing a patient).The method includes:

providing the CAR ligand (optionally, a labelled CAR ligand, e.g., a CARligand that includes a tag, a bead, a radioactive or fluorescent label);

acquiring the CAR-expressing cell (e.g., acquiring a sample containingCAR-expressing cells, such as a manufacturing sample or a clinicalsample);

contacting the CAR-expressing cell with the CAR ligand under conditionswhere binding occurs, thereby detecting the level (e.g., amount) of theCAR-expressing cells present. Binding of the CAR-expressing cell withthe CAR ligand can be detected using standard techniques such as FACS,ELISA and the like.

In another aspect, a method of expanding and/or activating cells (e.g.,immune effector cells) is disclosed. The method includes:

providing a CAR-expressing cell (e.g., a first CAR-expressing cell or atransiently expressing CAR cell);

contacting said CAR-expressing cell with a CAR ligand, e.g., a CARligand as described herein), under conditions where immune cellexpansion and/or proliferation occurs, thereby producing the activatedand/or expanded cell population.

In certain embodiments, the CAR ligand is present on a substrate (e.g.,is immobilized or attached to a substrate, e.g., a non-naturallyoccurring substrate). In some embodiments, the substrate is anon-cellular substrate. The non-cellular substrate can be a solidsupport chosen from, e.g., a plate (e.g., a microtiter plate), amembrane (e.g., a nitrocellulose membrane), a matrix, a chip or a bead.In embodiments, the CAR ligand is present in the substrate (e.g., on thesubstrate surface). The CAR ligand can be immobilized, attached, orassociated covalently or non-covalently (e.g., cross-linked) to thesubstrate. In one embodiment, the CAR ligand is attached (e.g.,covalently attached) to a bead. In the aforesaid embodiments, the immunecell population can be expanded in vitro or ex vivo. The method canfurther include culturing the population of immune cells in the presenceof the ligand of the CAR molecule, e.g., using any of the methodsdescribed herein.

In other embodiments, the method of expanding and/or activating thecells further comprises addition of a second stimulatory molecule, e.g.,CD28. For example, the CAR ligand and the second stimulatory moleculecan be immobilized to a substrate, e.g., one or more beads, therebyproviding increased cell expansion and/or activation.

In yet another aspect, a method for selecting or enriching for a CARexpressing cell is provided. The method includes contacting the CARexpressing cell with a CAR ligand as described herein; and selecting thecell on the basis of binding of the CAR ligand.

In yet other embodiments, a method for depleting, reducing and/orkilling a CAR expressing cell is provided. The method includescontacting the CAR expressing cell with a CAR ligand as describedherein; and targeting the cell on the basis of binding of the CARligand, thereby reducing the number, and/or killing, the CAR-expressingcell. In one embodiment, the CAR ligand is coupled to a toxic agent(e.g., a toxin or a cell ablative drug). In another embodiment, theanti-idiotypic antibody can cause effector cell activity, e.g., ADCC orADC activities.

Exemplary anti-CAR antibodies that can be used in the methods disclosedherein are described, e.g., in WO 2014/190273 and by Jena et al.,“Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to DetectCD19-Specific T cells in Clinical Trials”, PLOS March 2013 8:3 e57838,the contents of which are incorporated by reference.

In some aspects and embodiments, the compositions and methods herein areoptimized for a specific subset of T cells, e.g., as described in USSerial No. PCT/US2015/043219 filed Jul. 31, 2015, the contents of whichare incorporated herein by reference in their entirety. In someembodiments, the optimized subsets of T cells display an enhancedpersistence compared to a control T cell, e.g., a T cell of a differenttype (e.g., CD8+ or CD4+) expressing the same construct.

In some embodiments, a CD4+ T cell comprises a CAR described herein,which CAR comprises an intracellular signaling domain suitable for(e.g., optimized for, e.g., leading to enhanced persistence in) a CD4+ Tcell, e.g., an ICOS domain. In some embodiments, a CD8+ T cell comprisesa CAR described herein, which CAR comprises an intracellular signalingdomain suitable for (e.g., optimized for, e.g., leading to enhancedpersistence of) a CD8+ T cell, e.g., a 4-1BB domain, a CD28 domain, oranother costimulatory domain other than an ICOS domain. In someembodiments, the CAR described herein comprises an antigen bindingdomain described herein, e.g., a CAR comprising an antigen bindingdomain.

In an aspect, described herein is a method of treating a subject, e.g.,a subject having cancer. The method includes administering to saidsubject, an effective amount of:

1) a CD4+ T cell comprising a CAR (the CARCD4+) comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein;

a transmembrane domain; and an intracellular signaling domain, e.g., afirst costimulatory domain, e.g., an ICOS domain; and

2) a CD8+ T cell comprising a CAR (the CARCD8+) comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein;

a transmembrane domain; and

an intracellular signaling domain, e.g., a second co stimulatory domain,e.g., a 4-1BB domain, a CD28 domain, or another costimulatory domainother than an ICOS domain;

wherein the CARCD4+ and the CARCD8+ differ from one another.

Optionally, the method further includes administering:

3) a second CD8+ T cell comprising a CAR (the second CARCD8+)comprising:

an antigen binding domain, e.g., an antigen binding domain describedherein;

a transmembrane domain; and

an intracellular signaling domain, wherein the second CARCD8+ comprisesan intracellular signaling domain, e.g., a costimulatory signalingdomain, not present on the CARCD8+, and, optionally, does not comprisean ICOS signaling domain.

Biopolymer Delivery Methods

In some embodiments, one or more CAR-expressing cells as disclosedherein can be administered or delivered to the subject via a biopolymerscaffold, e.g., a biopolymer implant. Biopolymer scaffolds can supportor enhance the delivery, expansion, and/or dispersion of theCAR-expressing cells described herein. A biopolymer scaffold comprises abiocompatible (e.g., does not substantially induce an inflammatory orimmune response) and/or a biodegradable polymer that can be naturallyoccurring or synthetic. Exemplary biopolymers are described, e.g., inparagraphs 1004-1006 of International Application WO2015/142675, filedMar. 13, 2015, which is herein incorporated by reference in itsentirety.

Pharmaceutical Compositions and Treatments

In some aspects, the disclosure provides a method of treating a patient,comprising administering CAR-expressing cells produced as describedherein, optionally in combination with one or more other therapies. Insome aspects, the disclosure provides a method of treating a patient,comprising administering a reaction mixture comprising CAR-expressingcells as described herein, optionally in combination with one or moreother therapies. In some aspects, the disclosure provides a method ofshipping or receiving a reaction mixture comprising CAR-expressing cellsas described herein. In some aspects, the disclosure provides a methodof treating a patient, comprising receiving a CAR-expressing cell thatwas produced as described herein, and further comprising administeringthe CAR-expressing cell to the patient, optionally in combination withone or more other therapies. In some aspects, the disclosure provides amethod of treating a patient, comprising producing a CAR-expressing cellas described herein, and further comprising administering theCAR-expressing cell to the patient, optionally in combination with oneor more other therapies. The other therapy may be, e.g., a cancertherapy such as chemotherapy.

In an embodiment, cells expressing a CAR described herein areadministered to a subject in combination with a molecule that decreasesthe Treg cell population. Methods that decrease the number of (e.g.,deplete) Treg cells are known in the art and include, e.g., CD25depletion, cyclophosphamide administration, modulating GITR function.Without wishing to be bound by theory, it is believed that reducing thenumber of Treg cells in a subject prior to apheresis or prior toadministration of a CAR-expressing cell described herein reduces thenumber of unwanted immune cells (e.g., Tregs) in the tumormicroenvironment and reduces the subject's risk of relapse.

In one embodiment, a therapy described herein, e.g., a CAR-expressingcell, is administered to a subject in combination with a moleculetargeting GITR and/or modulating GITR functions, such as a GITR agonistand/or a GITR antibody that depletes regulatory T cells (Tregs). Inembodiments, cells expressing a CAR described herein are administered toa subject in combination with cyclophosphamide. In one embodiment, theGITR binding molecules and/or molecules modulating GITR functions (e.g.,GITR agonist and/or Treg depleting GITR antibodies) are administeredprior to the CAR-expressing cell. For example, in one embodiment, a GITRagonist can be administered prior to apheresis of the cells. Inembodiments, cyclophosphamide is administered to the subject prior toadministration (e.g., infusion or re-infusion) of the CAR-expressingcell or prior to apheresis of the cells. In embodiments,cyclophosphamide and an anti-GITR antibody are administered to thesubject prior to administration (e.g., infusion or re-infusion) of theCAR-expressing cell or prior to apheresis of the cells. In oneembodiment, the subject has cancer (e.g., a solid cancer or ahematological cancer such as ALL or CLL). In one embodiment, the subjecthas CLL. In embodiments, the subject has ALL. In embodiments, thesubject has a solid cancer, e.g., a solid cancer described herein.Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT PublicationNos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibodydescribed, e.g., in U.S. Pat. No. 7,025,962, European Patent No.:1947183B1, U.S. Pat. No. 7,812,135, U.S. Pat. No. 8,388,967, U.S. Pat.No. 8,591,886, European Patent No.: EP 1866339, PCT Publication No.: WO2011/028683, PCT Publication No.:WO 2013/039954, PCT Publication No.:WO2005/007190, PCT Publication No.: WO 2007/133822, PCT Publication No.:WO2005/055808, PCT Publication No.: WO 99/40196, PCT Publication No.: WO2001/03720, PCT Publication No.: WO99/20758, PCT Publication No.:WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No.7,618,632, and PCT Publication No.: WO 2011/051726.

In one embodiment, a CAR expressing cell described herein isadministered to a subject in combination with a GITR agonist, e.g., aGITR agonist described herein. In one embodiment, the GITR agonist isadministered prior to the CAR-expressing cell. For example, in oneembodiment, the GITR agonist can be administered prior to apheresis ofthe cells. In one embodiment, the subject has CLL.

The methods described herein can further include formulating aCAR-expressing cell in a pharmaceutical composition. Pharmaceuticalcompositions may comprise a CAR-expressing cell, e.g., a plurality ofCAR-expressing cells, as described herein, in combination with one ormore pharmaceutically or physiologically acceptable carriers, diluentsor excipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions can be formulated, e.g., forintravenous administration.

In one embodiment, the pharmaceutical composition is substantially freeof, e.g., there are no detectable levels of a contaminant, e.g.,selected from the group consisting of endotoxin, mycoplasma, replicationcompetent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residualanti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum,bovine serum albumin, bovine serum, culture media components, vectorpackaging cell or plasmid components, a bacterium and a fungus. In oneembodiment, the bacterium is at least one selected from the groupconsisting of Alcaligenes faecalis, Candida albicans, Escherichia coli,Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus pneumonia, and Streptococcuspyogenes group A.

When “an immunologically effective amount,” “an anti-cancer effectiveamount,” “a cancer-inhibiting effective amount,” or “therapeutic amount”is indicated, the precise amount of the compositions to be administeredcan be determined by a physician with consideration of individualdifferences in age, weight, tumor size, extent of infection ormetastasis, and condition of the patient (subject). It can generally bestated that a pharmaceutical composition comprising the immune effectorcells (e.g., T cells, NK cells) described herein may be administered ata dosage of 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to10⁶ cells/kg body weight, including all integer values within thoseranges. T cell compositions may also be administered multiple times atthese dosages. The cells can be administered by using infusiontechniques that are commonly known in immunotherapy (see, e.g.,Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

In some embodiments, a dose of CAR cells (e.g., CD19 CAR cells)comprises about 1×10⁶, 1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷,2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, or 5×10⁸ cells/kg. In some embodiments, adose of CAR cells (e.g., CD19 CAR cells) comprises at least about 1×10⁶,1.1×10⁶, 2×10⁶, 3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸,2×10⁸, or 5×10⁸ cells/kg. In some embodiments, a dose of CAR cells(e.g., CD19 CAR cells) comprises up to about 1×10⁶, 1.1×10⁶, 2×10⁶,3.6×10⁶, 5×10⁶, 1×10⁷, 1.8×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, or 5×10⁸cells/kg. In some embodiments, a dose of CAR cells (e.g., CD19 CARcells) comprises about 1.1×10⁶-1.8×10⁷ cells/kg. In some embodiments, adose of CAR cells (e.g., CD19 CAR cells) comprises about 1×10⁷, 2×10⁷,5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or 5×10⁹ cells. In someembodiments, a dose of CAR cells (e.g., CD19 CAR cells) comprises atleast about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, or5×10⁹ cells. In some embodiments, a dose of CAR cells (e.g., CD19 CARcells) comprises up to about 1×10⁷, 2×10⁷, 5×10⁷, 1×10⁸, 2×10⁸, 5×10⁸,1×10⁹, 2×10⁹, or 5×10⁹ cells.

In certain aspects, it may be desired to administer activated immuneeffector cells (e.g., T cells, NK cells) to a subject and thensubsequently redraw blood (or have an apheresis performed), activateimmune effector cells (e.g., T cells, NK cells) therefrom, and reinfusethe patient with these activated and expanded immune effector cells(e.g., T cells, NK cells). This process can be carried out multipletimes every few weeks. In certain aspects, immune effector cells (e.g.,T cells, NK cells) can be activated from blood draws of from 10 cc to400 cc. In certain aspects, immune effector cells (e.g., T cells, NKcells) are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60cc, 70 cc, 80 cc, 90 cc, or 100 cc.

The administration of the subject compositions may be carried out in anyconvenient manner. The compositions described herein may be administeredto a patient trans arterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally, e.g., by intradermalor subcutaneous injection. The compositions of immune effector cells(e.g., T cells, NK cells) may be injected directly into a tumor, lymphnode, or site of infection.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Example 1: Optimizing CART Production with Exogenous Cytokines

Cytokines have important functions related to T cell expansion,differentiation, survival and homeostasis. One of the most importantcytokine families for clinical use is the common γ-chain (γ_(c)) familycytokines, which includes interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15and IL-21 (Liao et al., 2013, Immunity, 38:13-25. IL-2 has been widelystudied as an immunotherapeutic agent for cancer. The supplement of IL-2enhanced the antitumor ability of anti-CD19 CAR-T cells in the clinicaltrials (Xu et al., 2013, Lymphoma, 54:255-60). However, theadministration of IL-2 is limited by side effects and a propensity forexpansion of regulatory T cells and the effect of activated induced celldeath (AICD) (Malek et al., 2010, Immunity, 33:153-65; and Lenardo etal., 1999, Annu Rev Immunol, 17:221-53). IL-7, IL-15, and IL-21 each canenhance the effectiveness of adoptive immunotherapies and seems to beless toxicity compared with IL-2 (Alves et al., 2007, Immunol Lett,108:113-20). Despite extensive preclinical and clinical studies on therole of the above cytokines, multi-parameter comparative studies on theroles of various exogenous γ_(c) cytokines on CAR-T cell adoptivetherapy are lacking.

Besides γ-chain cytokines, IL-18 is another immunostimulatory cytokineregulating immune responses, which enhances the production of IFN-γ by Tcells and augments the cytolytic activity of CTLs (Srivastava et al.,2010, Curr Med Chem, 17:3353-7). Administration of IL-18 is safe andwell tolerated, even when the dose reaching as high as 1000 μg/kg(Robertson et al., 2006, Clin Cancer Res, 12:4265-73). Therefore, IL-18could be another candidate used to boost the antitumor of CAR-T cells.

In this example, the effect of administration of different exogenouscytokines was examined for expansion, phenotype, in vitro effectorfunctions, and in vivo anti-tumor efficacy of T cells and folatereceptor alpha (FRα) CART cells.

The following materials and methods were used in the experimentsdescribed in this example.

CAR Construction and Lentivirus Preparation

The pELNS-C4-27z CAR vector was constructed as described previously(manuscript under review), Briefly, the pHEN2 plasmid containing theanti-FRα C4/AFRA4 scFv was used as a template for PCR amplification ofC4 fragment using the primers of 5′-ataggatcccagctggtggagtctgggggaggc-3′(SEQ ID NO: 19) and 5′-atagctagcacctaggacggtcagcttggtccc-3′ (SEQ ID NO:24) (BamHI and NheI were underlined). The PCR product and the thirdgeneration self-inactivating lentiviral expression vectors pELNS weredigested with BamHI and NheI. The digested PCR products were theninserted into the pELNS vector containing CD27-CD3z T-cell signalingdomain in which transgene expression is driven by the elongationfactor-1α (EF-1α) promoter.

High-titer replication-defective lentivirus was generated bytransfection of human embryonic kidney cell line 293T (293T) cells withfour plasmids (pVSV-G, pRSV.REV, pMDLg/p.RRE and pELNS-C4-27z CAR) byusing Express In (Open Biosystems) as described previously. Supernatantswere collected at 24 h and 48 h after transfection and concentrated byultracentrifugation. The virus titers were determined based on thetransduction efficiency of lentivirus to SupT1 cells by using limitingdilution method.

T Cells and Cell Lines

Peripheral blood lymphocytes were obtained from healthy donors afterinformed consent under a protocol approved by University InstitutionalReview Board at the University of Pennsylvania. The primary T cells werepurchased from the Human Immunology Core after purified by negativeselection. T cells were cultured in complete media (RPMI 1640supplemented with 10% FBS, 100 U/mL penicillin, 100 m/mL streptomycinsulfate) and stimulated with anti-CD3 and anti-CD28 mAbs-coated beads(Invitrogen) at a ratio of 1:1 following the instruction. Twenty-fourhours after activation, cells were transduced with lentivirus at MOI of5. Indicated cytokines were added to the transduced T cells from thenext day with a final concentration of 10 ng/mL. The cytokines werereplaced every 3 days.

The 293T cell used for lentivirus packaging and the SupT1 cell used forlentiviral titration were obtained from ATCC. The established ovariancancer cell lines SKOV3 (FRα+) and C30 (FRα−) was used as target cellfor cytokine-secreting and cytotoxicity assay. For bioluminescenceassays, SKOV3 was transduced with lentivirus to express fireflyluciferase (fLuc).

Flow Cytometric Analysis and Cell Sorting

Flow cytometry was performed on a BD FACSCanto. Anti-human CD45 (HI30),CD3 (HIT3a), CD8 (HIT8a), CD45RA (HI100), CD62L (DREG-56), CCR7(G043H7), IL-7Rα (A019D5), CD27 (M-T271), CD28 (CD28.2), CD95 (DX2),TNF-α (MAb11), IFN-γ (4S.B3), IL-2 (MQ1-17H12), perforin (B-D48),granzym-B (GB11) were obtained from Biolegend. Biotin-SP-conjugatedrabbit anti-human IgG (H+L) was purchased from Jackson Immunoresearchand APC conjugated streptavidin was purchased from Biolegand. Anti-humanBcl-xl (7B2.5) was purchased from SouhernBiotech. Apoptosis kit andTruCount tubes were obtained from BD Bioscience. For peripheral blood Tcell count, blood was obtained via retro-orbital bleeding and stainedfor the presence of human CD45, CD3, CD4 and CD8 T cells. HumanCD45+-gated, CD3+, CD4+ and CD8+ subsets were quantified with theTruCount tubes following the manufacturer's instructions.

In Vivo Study of Adoptive Cell Therapy

Female non-obese diabetic/severe combined immunodeficiency/γ-chain^(−/−)(NSG) mice 8 to 12 weeks of age were obtained from the Stem Cell andXenograft Core of the Abramson Cancer Center, University ofPennsylvania. The mice were inoculated subcutaneously with 3×10⁶ fLuc⁺SKOV3 cells on the flank on day 0. Four or Five mice were randomized pergroup before treatment. After tumors became palpable, human primary Tcells were activated and transduced as described previously. T cellswere expanded in the presence of IL-2 (5 ng/mL) for about 2 weeks. Whenthe tumor burden was ˜250-300 mm³, the mice were injected with 5×10⁶CAR-T cells or 100 μl saline intravenously and then received dailyintraperitoneal injection of 5 μg of IL-2, IL-7, IL-15, IL-18, IL-21 orphosphate buffer solution (PBS) for 7 days. Tumor dimensions weremeasured with calipers and tumor volumes were calculated with thefollowing formula: tumor volume=(length×width²)/2. The number andphenotype of transferred T cells in recipient mouse blood was determinedby flow cytometry after retro-orbital bleeding. The mice were euthanizedwhen the tumor volumes were more than 2000 mm³ and tumors were resectedimmediately for further analysis.

Statistical Analysis

Statistical analysis was performed with Prism 5 (GraphPad software) andIBM SPSS Statistics 20.0 software. The data were shown as mean±SEMunless clarified. Paired sample t-tests or nonparametric Wilcoxon ranktests were used for comparison of two groups and repeated measures ANOVAor Friedman test were used to test statistical significance ofdifferences among three or more groups. Findings were considered asstatistically significant when P-values were less than 0.05.

Results

1. Construction and Expression of Anti-FRα C4 CAR

The pELNS-C4-27z CAR comprised of the anti-FRα C4 scFv linked to a CD8αhinge and transmembrane region, followed by a CD3ζ signaling moiety intandem with the CD27 intracellular signaling motif (FIG. 1A). Primaryhuman T cells were efficiently transduced with C4 CAR lentiviral vectorswith transduction efficiencies of 43%˜65% when detected at 48 h aftertransduction (FIG. 1B). CAR expression levels were comparable betweenCD4+ and CD8+ T cells (52.6±10.2% vs. 49.5±17.1%, P=0.713).

2. Influence of Cytokines on Expansion of CAR Transduced T (CAR-T) Cell

The expansion and accumulation of CAR-T cells in the presence of variousγc cytokines and IL-18 was investigated. Three weeks after exposure tothe different cytokines in culture, CAR-T cells that had been culturedin the presence of IL2, IL-7 or IL-5 had expanded 1000-2000 fold. CAR-Tcells that had been cultured in the presence of IL-18, IL-21 or NC(control, no cytokine) demonstrated a less than 200 fold expansion (FIG.1C).

CAR expression levels were compared 15 days after transduction indifferent T cell populations: CD3+, CD4+, and CD8+ T cells. CARexpression levels of each group of CAR-T cells grown in the presence ofthe different cytokines were similar in CD3+, CD4+, and CD8+ T cellpopulations. CAR-T cells that had been cultured in the presence of IL-21showed higher levels of CAR expression than the CAR-T cells exposed toother cytokines (including IL-2 and the control cells that were notgrown in the presence of cytokines (NC) in the CD3+ and CD4+ T cellpopulations (FIG. 1D).

The reasons contributing to the higher accumulation of CAR-T cells wereanalyzed, specifically, proliferation and apoptosis of the T cells wasassessed. The proliferative response was measured by monitoring celldivision of CFSE labeled T cells cultured for 7 days. As shown in FIG.1E, T cells cultured with IL-2 and IL-15 showed the highestproliferative ability, followed by IL-7; while IL-21 and IL-18 were lesspotent mitogenic stimulants. Apoptosis of the T cells cultured in thedifferent cytokines was tested using Annexin-V staining. The resultsindicated that T cells cultured in IL-2, IL-7 and IL-15 underwent lessapoptosis when compared with NC, IL-18 and IL-21 groups (FIG. 1F). Next,the expression of Bcl-xL, a key negative regulator of lymphocyteapoptosis, was examined by FACs analysis. Consistent with the apoptosisresults in FIG. 1F, there is a trend that IL-2, IL-7 and IL-15 exposureresults in the up-regulation of Bcl-xL expression both in CD4+ and CD8+T cells, while IL-21 did not increase Bcl-xL expression (FIGS. 1G and1H). These results indicate that increased accumulation of T cellsexpanded in the presence of cytokines, e.g., IL-2, IL-7, or IL-15, maybe caused by both an increase in proliferation and a decrease inapoptosis by activation of the Bcl-xl anti-apoptotic pathway.

3. Influence of Cytokines on the Phenotypes of CAR-T Cells

Next, the phenotype of the CAR-T cells expanded in the presence ofexogenous cytokines was examined. The fresh T cells from healthy donorswere generally divided into four subsets based on CD45RA and CD62Lexpression: 1) naïve T cell (CD45RA+CD62L+, referred to as Tn), 2)central memory T cell (CD45RA−CD62L+, referred to as Tcm), 3) effectormemory T cell (CD45RA−CD62L−, referred to as Tem) and 4) CD45RA positiveeffector T cell (CD45RA+CD62L−, referred to as Temra). Then theexpression of CCR7, CD27, CD28, and CD95 are further evaluated for eachsubset (FIG. 2A). The latter three T cell subsets were positive for CD95while only small part of Tn expressed CD95 (3.6±1.4% in CD4+ and3.7±1.3% in CD8+ T cells). This small population also co-expressed CD27,CD28 and CCR7, and was considered as memory stem T cells (Tscm).However, after stimulation with anti-CD3/CD28 beads before and afterlentiviral transduction with CAR, CD95 was greatly up-regulated tonearly 100% in this population (FIG. 2B). The percentages ofCD45RA+CD62L+CD95+ T cells were greatly expanded after anti-CD3/CD28bead stimulation in both CD4+ and CD8+ T and CAR-T cells when comparedwith the fresh T cells (FIGS. 2C and 2D). This population highlyexpressed CD27, CD28 and CCR7 simultaneously (FIG. 2A), indicating itcould be defined as Tscm. Furthermore, CD8+ CAR-T cells had a higherpercentage of Tscm cells, which may be related to the higher proportionof Tn in initial CD8+ T cells (FIGS. 2F and 2G).

Fourteen days after co-culture with various cytokines, the proportion ofT cell subsets of CAR-T cells were investigated by measuring theexpression of CD45RA, CD62L and CD95. As shown in FIG. 2E, of the CD4+CAR-T cells, a significantly higher percentage of Tscm cells existed inthe IL-7 group compared with the IL-2 group, while the NC and IL-18groups presented lower percentages of Tscm but higher percentages ofTcm. The distribution of T cell subsets in the IL-15 group was similarwith the IL-2 group, while the IL-21 group presented a higher percentageof Tcm, while percentage of Tscm was comparable with the IL-2 group. TheCD8+ CAR-T cells demonstrated a similar trend as that of the CD4+ CAR-Tcells on the differentiation and distribution of the four T cell subsetsfor each cytokine-administered group, with higher proportions of Tscmcompared with CD4+ CAR-T cells in the corresponding group of CD8+ CAR-Tcells.

The abilities of various CAR-T cell subpopulations to self-renew and todifferentiate into other cell types were further studied. The foursubsets of CAR-T cells were sorted based on CAR, CD45RA and CD62Lexpression and cultured separately in medium containing IL-2 for 3 days.As shown in FIG. 2H, the Tscm subset was able to differentiate into allthe other three subsets, and Tcm and Temra subsets were able todifferentiate into Tem. These results indicate that CD62L+ and CD45RA+ Tcells were able to differentiate into CD62L- and CD45RA− T cells,respectively. The proliferation capacity of the four subsets wasassessed by CFSE dilution and then compared. The results showed the Tscmpresented stronger proliferation ability than other subsets (FIG. 2I).Furthermore, CD45RA expression inversely correlated with CFSE intensitywhile CD62L and CCR7 expression directly correlated with proliferation.In all cytokine groups, CD45RA+ T cells exhibited much lower CFSE levelsthan CD45RA dim and negative T cells (FIG. 3), indicating that CD45RA+ Tcells had stronger proliferation activity than CD45RA− T cells. Thus,the increased accumulation of T cells grown in the presence of IL-2,IL-7 and IL-15 may be related to the increased proportion of CD45RA+ Tcells (which have increased proliferation capacity) (FIG. 4B).

With regard to the phenotype of the CAR-T cells, CAR-T cells presentedlower expression of CD45RA, CD62L, CD27 and CD28, but higher expressionof CCR7 on the surface of T cells (FIG. 4A). The influence of cytokineson the phenotype of CAR-T cells were further assessed based on theexpression of the following surface markers: CD27, CD28, CD62L, CCR7 andIL7Ra. CAR-T cells grow in the presence of IL-18 showed quite similarexpression pattern with those grown without cytokine supplement. IL-2dramatically downregulated the expressions of CD27, CD28 CD62L, CCR7 andILR7α when compared with NC control. Of the other γc cytokines, comparedwith IL-2 exposed CAR-T cells, IL-7 exposed CAR-T cells presented higherCD62L, CD27 and CD28 expression but significantly decreased CCR7expression; IL-15 group CAR-T cells presented higher CD27 and CD28expression; and IL-21 exposed CAR-T cells presented higher CD62L, CCR7,CD27 and CD28 expression, indicating that IL-2 exposure induced theexpansion of a subset of T cells with a much more mature T cellphenotype than all other groups (FIG. 4B).

4. Influence of Cytokines on the Effector Function of CAR-T Cells

To investigate the influence of cytokines on CAR-T cell effectorfunction, the cytokine production capability of CAR-T cells afterstimulation with FRα-expressing SKOV3 cells was assessed. Following 5hours stimulation, TNF-α, IFN-γ and IL-2 were detectable in thecytoplasm of CAR-T cells, with 41.5-54.0% of the CAR-T cells producedTNF-α, 12.4-15.3% of the CAR-T cells produced IFNγ, and 4.3-6.5% ofCAR-T cells produced and IL-2 (FIGS. 5A&B). IL-2, IL-7 and IL-15exposure during expansion promoted CAR-T cells to produce TNF-α, whilethe numbers of IFN-γ and IL-2 producing CAR-T cells were comparableamong all the cytokine groups (FIGS. 5B, 5C, and 5D). Next, thefractions of responding CAR-T cells and their polyfunctionality werecompared (FIG. 5E). In comparison to exposure to IL-2 during expansion,exposure to IL-18, IL-21 or no cytokine exposure during expansioninduced less cytokine-producing CAR-T cells, and less CAR-T cellspossessed the ability to produce multiple cytokines when stimulated bytarget cells. These results are consistent with the phenotype that theCAR-T cells in IL-18, IL-21 and NC groups were less differentiated thanthose in the IL-2 exposed group.

Then, the effect of cytokine exposure during expansion on the expressionof the cytolytic molecules perforin and granzyme-B after antigenstimulation was determined (FIG. 5I). Similar with TNF-α production, theCAR-T cells exposed to IL-2, IL-7, and IL-15 demonstrated increasedperforin expression compared with CAR-T cells exposed to NC, IL-18 andIL-21. However, although CAR-T cells exposed to IL-21 produce less TNF-αand perforin, they produced the highest level of granzyme-B. The nexthighest levels of granzyme-B production were observed in CAR-T cellsexposed to IL-2 and IL-15 during expansion. CAR-T cells in IL-18 grouppresented the least amount of both perforin and granzyme-B expressionafter antigen stimulation (FIGS. 5G and 5H).

Finally, the tumor lysis activity by CAR-T cells exposed to variouscytokines during exposure was quantified by luciferase assay. As shownin FIG. 5F, CAR-T cells co-cultured with IL-2 and IL-15 lysed the SKOV3more efficiently than those with NC and IL-18 (both P<0.05).

The association between phenotype of the CAR-T cells and their functionwas further confirmed. The T cells 14 days were sorted after lentiviraltransduction based on CAR and CD62L expression. The CD62L+CAR-T cells(Tscm and Tcm) exhibited less cytokine production activity and weakercytolytic capacity when compared with CD62L− CAR-T cells (Tem and Temra)(FIG. 6). In this perspective, CAR-T cells exposed to IL-2 and IL-15produced more cytokines and presented stronger tumor lysis activity,which might be partially attributed to the higher proportions of Tem andTemra in these groups.

5. Expansion and Phenotype of CAR-T Cells after Antigen Challenge

To investigate the influence of cytokines on CAR-T cell expansion underthe challenge of specific antigen, the CAR-T cells exposed to IL-2 fortwo weeks were co-cultured with SKOV3 (FRα+) or C30 (FRα−) cells in thepresence of indicated cytokines for 7 days. Similar to the antigen-freecircumstance, CAR-T cells exposed to IL-2, IL-7 and IL-15 presentedhigher fold expansion than CAR-T cells exposed to other cytokines. TheCAR-T cells exposed to IL-21 during expansion were more likely toundergo apoptosis (FIG. 7A). However, when the CAR-T cells exposed tothe indicated cytokines for two weeks were co-cultured with SKOV3 or C30cells without further cytokine supplement for 7 days, the accumulationof CAR-T cells were comparable among all groups, with those having beenexposed to IL-15 and IL-18 undergoing the least amount of apoptosis(FIG. 7B). The T cells with the highest CAR expression was the groupexposed to IL-21 during expansion (FIG. 7C). The phenotypes of CAR-Tcells were also analyzed. CD27 and CD28 were highly expressed in CD8+and CD4+CAR-T cells in all cytokine groups, respectively. Similar toantigen-free expansion, IL-21 exposure increased the expression of CD27and CD28 in CAR-T cells (FIG. 7D). As to the four subsets of memory Tcells, the results were different from antigen-free study: Tscm was rareand Tem accounted for more than 50% in no cytokine, IL-18 and IL-21 allgroups. Cytokines had no significant impact on the composition of memoryT subsets and IL-7 exposure did not favor the increase of Tscm (FIG.7E).

6. Anti-Tumor Efficacy of Various Cytokines in Animal Models

To evaluate the effects of various cytokines during ex vivo expansion ofCAR-T cells on the efficacy of CAR-T cells in vivo, the persistence ofCAR-T cells and outcome was investigated by using a NSG mouse xenograftmodel of ovarian cancer. Mice bearing subcutaneous SKOV3 tumors wereintravenously injected with two doses of 5×106 C4-27z CAR-T cells whichhad been exposed to the indicated cytokines ex vivo for 2 weekspreviously (FIG. 8A). All mice receiving C4-27z CAR-T cell infusionpresented less tumor burden when compared with those injected withuntransduced T cells and anti-CD19 CAR-T cells (FIGS. 8B&8C). Of thevarious cytokine groups, mice receiving CAR-T cells with previous IL-2exposure showed the highest tumor burden, consistent with the leastamount of circulating human T cell in these mice. The tumors in NC,IL-7, IL-15, IL-18 and IL-21 groups were all significantly suppressed oreven disappeared, without any statistical difference on tumor size. Thepersistence of transferred T cells in the peripheral blood wasdetermined 15 and 32 days after adoptive transfer. Mice receiving IL-7and IL-21 treated CAR-T cells seemed to have higher amount of human Tcells than other groups in the peripheral blood on day +15, while micereceiving IL-2 treated CAR-T cells had the lowest number of human Tcells (FIG. 8D). As to the percentages of different CAR-T cellpopulations, NC, IL-15, IL-18 and IL-21 exposed groups all presentedhigher CD4+ CAR-T cells when compared with IL-2 group, while thepercentages of CD8+ CAR-T cells were comparable among all the groups(FIG. 8E). Of the T cell phenotypes, CD62L, CD27 and CD28 were expressedonly on about 5-10% of T cells and were comparable among all groups,except that CD8+ T cells in IL-21 group expressed higher CD28 than thosein IL-2 and NC group (both P<0.05) (FIGS. 8F&8G). Interestingly, NC andIL-18 had a much higher amount of CD45RA+CD62L+ T cells in the blood,which was opposite to the result obtained from the in vitro experiment(FIGS. 8F, 2E&2F). On day +32, the circulating human T cells in allCAR-T cell groups expanded significantly except the IL-2 group, with anaverage T cell account of 14907/μl to 19651/μl (and only 242/μl in theIL-2 group). Two mice died although the tumors were regressed.

Discussion

IL-2 is the most frequently used cytokine for generating lymphocytes foradoptive immunotherapy. It promotes T cell survival and expansion,enhances tumor-killing ability of T cells. However, the action of IL-2is limited as it results in activation induced cell death (AICD) ofT-cell and the development of regulatory T-cell (Malek et al., Immunity,2010, 33:153-65; and Lenardo et al., Annu Rev Immunol, 1999, 17:221-53).In this example, IL-2 significantly increased the accumulation of CAR-Tcells and their cytotoxicity ability, but IL-2 exposed CAR-T cellspresented inferior antitumor immunity in vivo following adoptivetransfer. This finding demonstrates an inverse relationship between invitro tumor-lysis and in vivo tumor eradication. IL-2 exposed CAR-Tcells displayed a relative mature phenotype with low expression ofCD62L, CCR7, CD27 and CD28, which are less persistent in vivo (Yang etal., Cancer Immunol Immunother, 2013, 62:727-36). Recent studies haveindicated that adoptive transfer of less differentiated T cellscorrelates with superior tumor regression, which supports the findingthat IL-2 exposed CAR-T cells are less effective than other group(Gattinoni et al., Nat Med, 2011, 17:1290-7; and Markley et al., Blood,2010, 115:3508-19).

IL-15 presented similar performance of stimulating CAR-T cell expansionand tumor-lysis function as IL-2, but induced a less differentiatedphenotype (higher expression of CD27 and CD28). Therefore, IL-15supports the persistence of CAR-T cells in vivo and shows betterantitumor immunity in animal models.

Compared with IL-2 and IL-15, IL-7 showed similar capability to promoteCAR-T cell expansion, but induced higher level of CD62L expression andexhibited the highest proportion of CAR-Tscm cells in an antigen-freecircumstance. Therefore, compared to CAR-T cells exposed to IL-2, exvivo exposure of IL-7 without antigen challenge enhanced the antitumorefficacy of the CAR-T cells. IL-7 exposed CAR-T cells did not result inbetter in vivo antitumor efficacy than IL-2, and efficacy was inferiorto IL-15 due to the less expansion of CAR-T cells under antigenchallenge.

IL-21 exerted few effects on CAR-T cell accumulation as it could notenhance anti-apoptosis ability, e.g., by promoting Bcl-xL expression.However, IL-21 induced the expansion of less differentiated CAR-T cells,with a phenotype of high expression of CD62L, CCR7, CD27 and CD28, evenunder the circumstance of antigen challenge. Therefore, IL-21 exposedCAR-T cells showed best persistence in animal models and IL-21 injectionin vivo, and also presented a better efficacy in promoting tumoreradication than other cytokine groups except IL-15. These results areconsistent with previous finding that less differentiated CAR-T cellscorrelates with superior tumor regression.

IL-18 is proinflammatory cytokine belonging to the IL-1 family, whichregulates both innate and adaptive immune responses by activatingmonocytes, NK cells, and T cells and production of IFN-γ as well asother cytokines in vivo (Srivastava et al., Curr Med Chem, 2010,17:3353-7). The results presented herein indicates that IL-18 has littleimpact on CAR-T cell's expansion, phenotype and function in ex vivoexperiments, as most of the results in IL-18 groups are similar andcomparable with NC group. IL-18 promoted little proliferation of T cellsand maintained more T cell survival under antigen challenge compared tothe control (NC) group. In vivo studies show that IL-18 has nosignificant impact on CAR-T cell efficacy when compared with micewithout cytokine supplement.

In summary, the findings of these experiments indicate that IL-2supplement ex vivo for CAR-T cell expansion is not an optimal strategyalthough it is widely used. As to IL-18, IL-21 or no cytokinesupplement, although they may induced relative effective CAR-T cells,they do not promote CAR-T cell expansion effectively enough, such thatenough CAR-T cells could be prepared for clinical use in a limitedexpansion time. Therefore, IL-15 and IL-7 may be better agents for CAR-Tcell expansion. Furthermore, the combination of IL-7 and IL-15supplement instructs the generation of Tscm, which is beneficial toproduce more “young” CAR-T cells. As to in vivo cytokine injection, allγc cytokines supplement enhance antitumor efficacy, as many of themfavor the expansion of CAR-T cells, with IL-15 presenting best effect.Mice receiving IL-15 exposed CAR-T cells by injection had increasedefficacy, due in part to the increased expansion ability and increasedpersistence of the CAR-T cells during tumor treatment. Thus, the resultsof these experiments indicate that IL-7 and IL-15 show promise topromote CAR-T cell expansion and induce T cell phenotypes that are mostefficacious for therapeutic treatment.

Example 2: Effect of CD25 Depletion on Cell Growth and TransductionEfficiency

The interleukin-2 a-chain, also known as CD25, is expressed byregulatory T cells (Tregs) but has also been observed on chronic B cellleukemia (CLL) cells (in greater than 85% of CLL patients). Tregs haveimmune suppressing functions and can impede the efficacy ofimmunotherapy, e.g., by inhibiting T cell proliferation. Currentisolation or enrichment of T cells from CLL patients by apheresisusually contains a significantly increased proportion of Tregs as wellas CLL cells. The depletion of Tregs and CLL cells in the startingmaterial by CD25 depletion methods may significantly improve the purityof effector T cells, and thereby increase the potency of CAR19expressing T cells, e.g., CART19 cells.

FIG. 9A shows flow cytometry analysis plots of the cells from anapheresis of a CLL patient. Cells were first sorted for CD45 expression,and the CD45-expressing (CD45+) subset was then further analyzed forCD25 and CD3 expression. As shown in the panel on the right of FIG. 9A,CLL patients exhibit a high percentage of CD25 expressing cells.

Optimizing CD25 Depletion

A validation experiment was performed to identify the optimal conditionsfor CD25 depletion from the aphereses from two patients using CD25Reagent from Miltenyi in a CliniMACS System. CD25 depletion reagent wasused at 100%, 70%, and 30% of the manufacturer's recommended amount toidentify whether the same depletion efficiency could be obtained byusing less reagent. Two different tubing sets from Miltenyi were alsotested. The depletion was performed in accordance with themanufacturer's directions. The results from the experiments are shown inthe table below. For control, selection using anti-CD3/CD28immunomagnetic beads was performed.

TABLE 5 Experimental results from CD25 depletion. CD25 depletion arms 100%   70%   30% Miltenyl tubing set 161-01 CliniMACS programENRICHMENT1.1 Patient cells UPCC04409-15 % CD45+CD25+ cells 83.56%  %CD45+CD3+ cells 8.66% % CD45+CD3+CD25− cells 5.70% #CD25+ cells totarget  2.E+09  2.E+09  2.E+09 #apheresed cells for CD25 depletion2.39E+09 3.41E+09 7.97.E+09  CD25 bead volume used (mL) 2.5 2.5 2.5Cell# in CD25−depleted fraction 1.05E+09 1.86E+09 3.36E+09 Cell# inCD25−enriched fraction 2.05E+08 2.58E+08 5.19E+08 Expected CD25− T-cellyield 1.36E+08 1.95E+08 4.54E+08 % T cells in depleted fraction 6.26%4.08% 2.50% Observed yield CD25− T cells 6.57E+07 7.55E+07 8.40E+07Yield of CD3+CD25− as % of expected   48%   39%   18% % B cells indepleted fraction 90.50%  91.6% 95.30%  Viability CD25+ fraction 94.4%96.2% 91.1% Viability CD25− fraction 95.8% 95.0% 99.0%The expected CD25− (CD25-negative) T cell yield represents thecalculated CD25− T cell yield calculated by assuming 100% efficiency inthe respective manipulations. The observed yield of CD25− T cellsrepresents the number of CD25− T cells after the respectivemanipulations. As shown in Table 5, using less reagent than recommendedby the manufacturer did not result in the same efficiency in CD25depletion. Using different tubing resulted in an increase in T cellenrichment by one log.

FIG. 9B shows representative flow cytometry analysis plots (top panels)demonstrating the efficiency of CD25 depletion compared to the totalcells from the apheresis (top left panel of FIG. 9B), control CD3/CD28selected cells (top middle left panel of FIG. 9B), CD25 depleted cells(top middle right panel of FIG. 9B), and CD25 enriched cells (top rightpanel of FIG. 9B). The histograms (bottom panels) show the monocytecontent of the cell population, as determined by CD14 expression of theCD3-CD19− subset. These results indicate efficient CD25 depletion andthat CD25 depletion also resulted in significant monocyte content (61.1%CD14-expressing cells compared to less than 2% in the total cells fromapheresis, control, and the CD25 enriched cells.

Effect of CD25 Depletion on T Cell Population and Proliferation

Next, the quality of the T cell product after CD25 depletion wasassessed by determining the proportion of CD4+ and CD8+ T cells andproliferation capacity.

To determine the proportion of specific T cells populations, cells wereanalyzed by flow cytometry nine days after selection by anti-CD3/CD28 orCD25 depletion as described above. The results show thatCD3/CD28-selected T cells had a greater proportion of CD4+ T cellscompared to CD25 depleted cells (84.6% compared to 46.8% CD4+ T cells,boxes at the top left of the panels in FIG. 10). Conversely, CD25depleted cells had a greater proportion of CD8+ T cells compared to theCD3/CD28-selected cells (47.2% compared to 11.5% CD8+ T cells; boxes atthe bottom right of the panels in FIG. 10). Therefore, CD25 depletionresults in T cells with a greater proportion of CD8+ T effector cells.

Proliferation capacity and cell viability was also assessed in control(CD3/CD28 selected cells) and CD25 depleted cells. 1.6×10⁷ cells fromcontrol and CD25 depleted cells were plated and the cell number andviability was determined over 10-13 days. FIG. 11A shows the total cellnumber over time and FIG. 11B shows the calculated population doublings(calculated from the total number of cells). The results indicate thatthe CD25 depleted cells demonstrated similar growth characteristics tothe control cells. FIG. 11C shows the percentage of viable cells, andthe results show that viability was also similar between control andCD25 depleted cells.

Effect of CD25 Depletion on Lentiviral Transduction Efficiency

The effect of CD25 depletion on lentiviral transduction efficiency wasassessed by determining the expression of CAR after transduction. Apatient apheresis was depleted with CD25 cells as described above. Theefficiency of the CD25 depletion is demonstrated in the flow cytometryanalysis plots comparing the CD25-expressing population before(apheresis sample) and after CD25 depletion (CD25-depleted fraction)(FIG. 12A). After CD25 depletion, the CD25 depleted fraction containedabout 59.2% of CD25 negative cells and only 10.3% CD25 positive cells.

The CD25 depleted fraction was transduced with a lentiviral constructencoding CAR19. After 11 days of culture, CAR expression was assessed byflow cytometry. Cells that were untransduced and transduced CD3 selectedcells were used as controls. As shown in FIG. 12B, CAR19 expression wassignificantly higher in CD25 depleted cells compared to CD3 selectedcells (51.4% compared to 12.8%). This result demonstrates that CD25depleted cells have improved lentiviral transduction efficiency, whichmay be important for improved therapeutic effect in CART therapy.

Example 3: Using Cytokines with CD25-Depleted Cells

In this example, the effect of CD25 depletion with cytokine supplementduring expansion in culture was examined Peripheral blood mononuclearcells (PBMCs) were isolated from a patient and were either leftunmanipulated or were depleted of CD25-expressing cells as described inExample 2. T cell enrichment was achieved by stimulation with anti-CD3and CD28 coated beads. The T cells were immediately cultured in mediasupplemented with 10 ng/ml IL-7, 10 ng/ml IL-15, or the combination of10 ng/ml IL-7 and 10 ng/ml IL-15. At day 3, medium was changed with thesame cytokines added. At day 5, the medium containing 100 IU IL-2/ml wasadded, and the cells were grown for a total of 10 days.

Flow cytometric analysis shows the change in distribution of CD3 andCD19 cells in CD25 depleted cells compared to unmanipulated PBMC(standard CD3/CD28 selection) after culture in the presence of IL7,IL-15, or IL7 and IL15. The distribution of CD3, CD19, and CD25expressing cells in the starting population (e.g., before CD25 depletionand before culture with cytokine supplementation) is shown in FIG. 13.The starting population had a high proportion of CD3-CD19+ cells (toppanel, FIG. 11) and a high proportion of CD25-expressing cells (bottompanels, FIG. 13). After manipulation (CD25 depletion) and culture withcytokines, the distribution changed as shown in FIG. 14. CD25 depletedcells overall showed greater reduction in CD19-expressing cells comparedto the unmanipulated cells.

Proliferation capacity was also assessed for the same cell samples bydetermining the total number of cells in culture at day 10 afterstimulation with anti-CD3 and anti-CD28 coated beads. The cell numbersfor each cell sample are shown below and in FIG. 15.

TABLE 6 In vitro expansion Cells Cytokines added # Cells in cultureUnmanipulated IL-7 1.24 × 10⁶ IL-15 0.92 × 10⁶ IL-7 + IL-15 0.52 × 10⁶CD25-depleted IL-7 0.93 × 10⁶ IL-15 1.95 × 10⁶ IL-7 + IL-15 3.03 × 10⁶These results show that supplementation of IL-15 during culture of CD25depleted T cells resulted in increased expansion compared tounmanipulated cells. Addition of IL-7 and IL-15 in the media duringculture resulted in significant increase in expansion compared tounmanipulated cells, and compared to adding the cytokines IL-7 or IL-15independently. Thus, the combination IL-7 and IL-15 supplement resultedin T cells with the most increased proliferation capacity.

Example 4: Three-Day Manufacturing Process

With the use of gene transfer technologies, T cells can be geneticallymodified to stably express antibody binding domains on their surfacethat endow the T cells with specificities that are independent of theconstraints imposed by the major histocompatibility complex (MHC). CARtherapies can utilize synthetic proteins expressed on T-cells that fusean antigen recognition fragment of an antibody (an scFv, or single-chainvariable region fragment) with an intracellular domain of the CD3-zetachain. Upon interaction with a target cell expressing the scFv's cognateantigen, CARs expressed on T cell cells can trigger T-cell activationleading to target cell killing (also referred to as target cell lysis).When combined with additional costimulatory signals such as thecytoplasmic domain of CD137 or CD28, these receptors can also stimulateT cell proliferation and increase CAR-modified T cell (CART) persistencein vivo.

The mechanisms underlying CART persistence are being explored. Signalingdomains in CARs appear to be important factors determining persistence;however, a number of published studies in mice and non-human primates(Klebanoff, C. A., et al. (2005). “Central memory self/tumor-reactiveCD8+ T cells confer superior antitumor immunity compared with effectormemory T cells.” Proc Natl Acad Sci USA 102(27): 9571-9576, Berger, C.,et al. (2008). “Adoptive transfer of effector CD8+ T cells derived fromcentral memory cells establishes persistent T cell memory in primates.”J Clin Invest 118(1): 294-305, Hinrichs, C. S., et al. (2009).“Adoptively transferred effector cells derived from naive rather thancentral memory CD8+ T cells mediate superior antitumor immunity.” ProcNatl Acad Sci USA 106(41): 17469-17474, Wang, X., et al. (2011).“Engraftment of human central memory-derived effector CD8+ T cells inimmunodeficient mice.” Blood 117(6): 1888-1898) and data from humanclinical trial correlative studies suggest that the phenotype of T cellsin the adoptively transferred product is also an important determinantof T cell persistence following adoptive transfer (Xu, Y., et al.(2014). “Closely related T-memory stem cells correlate with in vivoexpansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15.”Blood 123(24): 3750-3759). While not wishing to be bound by theory, itis hypothesized that the T cells expanded during ex vivo culture underthe commonly employed system of anti-CD3 and anti-CD28 agonistantibodies combined with exogenous IL-2 biases the expanded T cellstowards more differentiated cells with reduced persistence. It washypothesized that replacing IL-2 with IL-7 and IL-15 would bias ex vivoculture over time towards cells with a more optimal Tscm/cm phenotype.It was also hypothesized that products with less ex vivo culture mighthave a greater proportion of cells with the optimal phenotype forengraftment and persistence following adoptive transfer.

This Example both characterizes the phenotype of cells over time inculture and evaluates the functionality of cells at different timepoints following activation and transduction.

Bead removal from T cells following activation with CD3/28-stimulatorybeads can be accomplished with high efficiency by mechanical disruptionas shown in FIGS. 27A-27D.

Bead detachment was accomplished in these preclinical studies by repeatpassage (extrusion) of the cell suspension through a narrow bore pipettip. In a closed culture system, extrusion to create gentle shearingcould be performed using a closed system that incorporates a set ofnarrow bore tube in a closed system prior to bead removal.

As shown in FIG. 28, T cells that are activated, transduced with aCD19-specific CAR bearing BBz signaling and harvested from T cellscultures at time points as early as day 3 following activation havepotent, antigen-specific cytotoxic activity in vitro. These earlyharvest cells are also able to produce cytokines with a similar patternand quantity to those cultured for longer periods of 5 or 9 days ex vivo(FIG. 29A) with similar basal secretion (FIG. 29B).

During ex vivo culture, the population of T cells undergoes aprogressive transition towards a more differentiated phenotype with anincreasing proportion of T effector memory (Tem) and Tcm cells as shownin FIG. 30A. This increase in proportion of Tem and Tcm cells resultsfrom an increase in more differentiated T cell subsets with minimalexpansion and/or decrease in the naive-like/Tscm cells (FIG. 30B).

FIG. 31A shows the experimental setup for testing the potency of CARTcells produced under different conditions. FIG. 31B shows the potency ofIL2-treated CART cells in slowing Nalm6 tumor growth in mice. Day 3cells demonstrate enhanced potency at all doses. T cells activated andtransduced with a CD19-specific CAR bearing the 4-1BBz signaling domainshow more potent in vivo anti-leukemic activity when harvested at theearly time points. This increased potency is demonstrated by the lowerdoses of T cells required to eradicate leukemia with day 3 harvestedCART19 cells compared with day 5 or day 9 harvested cells. Proportionsof CAR+ cells are shown in FIG. 31C.

Cells treated with IL-2 and IL7/15 were harvested in two different days(3, 9). A relatively low dose of 0.7e6 CAR+ cells was used. T cellsactivated, transduced with a CD19-specific CAR bearing the 4-1BBzsignaling domain and cultured in IL-7/IL-15 showed more potent activityat limiting doses than cells expanded in IL-2 at early (day 3) and late(day 9) time points (FIG. 31D). Day 3 cells treated with IL7/15demonstrate enhanced potency at early time points.

EQUIVALENTS

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific aspects, it is apparent that other aspects and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such aspects andequivalent variations.

What is claimed is:
 1. A method of making a population of chimericantigen receptor (CAR)-expressing immune effector cells, the methodcomprising: (a) providing a population of cells comprising immuneeffector cells from an apheresis sample from a patient with chroniclymphocyticleukemia; (b) depleting CD25-expressing cells from thepopulation of cells of step (a) to provide a population of CD25-depletedcells, wherein the population of CD25-depleted cells is a populationthat contains less than 50% CD25+ cells in the population of cells ofstep (a) when assessed by flow cytometry; (c) contacting the populationof CD25-depleted cells with anti-CD3 and anti-CD28 coated beads; and (d)transducing the population of CD25-depleted cells with a lentivirusvector encoding a chimeric antigen receptor; to thereby provide apopulation of CAR-expressing immune effector cells.
 2. The method ofclaim 1, wherein the population of CD25-depleted cells is a populationthat contains less than 30% CD25+ cells.
 3. The method of claim 1,wherein the population of CD25-depleted cells is a population thatcontains less than 20% CD25+ cells.
 4. The method of claim 1, whereinthe population of CD25-depleted cells is a population that contains lessthan 10% CD25+ cells.
 5. The method of claim 1, further comprising astep of depleting cells that express a tumor antigen prior to thecontacting step.
 6. The method of claim 1, further comprising a step ofdepleting cells that express a check point inhibitor prior to thecontacting step.
 7. The method of claim 1, wherein the CD25-expressingcells are depleted by contacting the population of cells from theapheresis sample with anti-CD25 antibody coated beads.
 8. A populationof chimeric antigen receptor expressing immune effector cells producedby the method of claim
 1. 9. A population of chimeric antigen receptorexpressing immune effector cells produced by the method of claim
 5. 10.A population of chimeric antigen receptor expressing immune effectorcells produced by the method of claim
 6. 11. A population of chimericantigen receptor expressing immune effector cells produced by the methodof claim
 7. 12. A method of making an expanded population of chimericantigen receptor (CAR)-expressing immune effector cells, the methodcomprising: (a) providing a population of cells comprising immuneeffector cells from an apheresis sample from a patient with chroniclymphocytic leukemia; (b) depleting CD25-expressing cells from thepopulation of cells of step (a) to provide a population of CD25-depletedcells, wherein the population of CD25-depleted cells is a populationthat contains less than 50% CD25+ cells in the population of cells ofstep (a) when assessed by flow cytometry; (c) contacting the populationof CD25-depleted cells with anti-CD3 and anti-CD28 coated beads; (d)transducing the population of CD25-depleted cells with a lentivirusvector encoding a chimeric antigen receptor; and (e) culturing thechimeric antigen receptor expressing immune effector cells in culturemedia supplemented with IL-15 or a combination of IL-15 and IL-7 for aperiod of between 3 and 9 days; to thereby provide an expandedpopulation of CAR-expressing immune effector cells.
 13. The method ofclaim 12, wherein the culturing period is less than 6 days.
 14. Themethod of claim 12, wherein the culturing period is less than 5 days.15. The method of claim 12, wherein the culturing period is less than 4days.
 16. An expanded population of chimeric antigen receptor expressingimmune effector cells produced by the method of claim
 12. 17. Anexpanded population of chimeric antigen receptor expressing immuneeffector cells produced by the method of claim
 15. 18. A method ofmaking an expanded population of CD19 chimeric antigen receptor (CD19CAR)-expressing immune effector cells, the method comprising: (a)providing a population of cells comprising immune effector cells from anapheresis sample from a patient with chronic lymphocytic leukemia; (b)depleting CD25-expressing cells from the population of cells to providea population of CD25-depleted cells, wherein the population ofCD25-depleted cells is a population that contains less than 50% CD25+cells in the population of cells of step (a) when assessed by flowcytometry; (c) contacting the population of CD25-depleted cells withanti-CD3 and anti-CD28 coated beads; (d) transducing the population ofCD25-depleted cells with a lentivirus vector encoding a CD19 CAR; and,(e) culturing the chimeric antigen receptor expressing immune effectorcells in culture media supplemented with IL-15 or a combination of IL-15and IL-7 for a period of between 3 and 9 days; to thereby provide anexpanded population of CD19 CAR-expressing immune effector cells. 19.The method of claim 18, wherein the culturing period is less than 4days.
 20. An expanded population of CD19 chimeric antigen receptorexpressing immune effector cells produced by the method of claim
 18. 21.The method of claim 1, further comprising expanding the population ofCD25-depleted cells.
 22. The method of claim 21, wherein the populationof CD25-depleted cells is expanded in the presence of a cytokine for aperiod of 8 days or less.
 23. The method of claim 1, wherein thepopulation of CD25-depleted cells is a population that contains lessthan 50%, 30%, 20%, or 10% of CD25-depleted tumor cells.
 24. The methodof claim 1, wherein the population of CAR-expressing immune effectorcells expresses a CAR that specifically binds to CD19.
 25. The method ofclaim 24, wherein the population of CAR-expressing immune effector cellsexpresses a CAR comprising an antigen binding domain comprising a lightchain variable domain (VL) and a heavy chain variable domain (VH) havingthe amino acid sequence of SEQ ID NO: 40 or SEQ ID NO:
 51. 26. Themethod of claim 12, wherein the population of CAR-expressing immuneeffector cells expresses a CAR that specifically binds to CD19.
 27. Themethod of claim 26, wherein the population of CAR-expressing immuneeffector cells expresses a CAR comprising an antigen binding domaincomprising a light chain variable domain (VL) and a heavy chain variabledomain (VH) having the amino acid sequence of SEQ ID NO: 40 or SEQ IDNO:
 51. 28. The method of claim 18, wherein the population ofCAR-expressing immune effector cells expresses a CAR comprising anantigen binding domain comprising a light chain variable domain (VL) anda heavy chain variable domain (VH) having the amino acid sequence of SEQID NO: 40 or SEQ ID NO: 51.